Brain derived and glial cell line derived neurotrophic factor fusion protein immobilization to laminin

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1 178 Brin derived nd glil ell line derived neurotrophi ftor fusion protein immoiliztion to lminin BAOXIN WANG 1*, JUNJIE YUAN 2*, JIAFENG XU 3, XINWEI CHEN 1, XINJIANG YING 1 nd PIN DONG 1 1 Deprtment of Otolryngology, Hed nd Nek Surgery, Shnghi Jio Tong University Affilited to Shnghi First People's Hospitl, Shnghi ; 2 Deprtment of Orthopedis, Shnghi Fengxin Distrit Centrl Hospitl, Shnghi Jio Tong University Affilited to Sixth People's Hospitl South Cmpus, Shnghi ; 3 Shool of Eonomis nd Finne, Shnghi Interntionl Studies University, Shnghi , P.R. Chin Reeived July 8, 2015; Aepted Septemer 1, 2016 DOI: /etm Astrt. Dmge to the reurrent lryngel nerve often uses horseness, dyspne, dysphgi, nd sometimes sphyxi due to vol ord prlysis whih result in redution of qulity of life. Brin derived neurotrophi ftor (BDNF) nd glil ell line derived neurotrophi ftor (GDNF) ply ritil roles in peripherl nerve regenertion. However, methods for effiiently delivering these moleules re lking, whih limits their use in linil pplitions. The present study reports n effetive strtegy for trgeting BDNF nd GDNF to lminin y fusing the N terminl domins of these moleules with grin (NtA). More speifilly, lminin inding effiy ws ssessed nd sustined relese ssys of the delivery of BDNF or GDNF fused with NtA (LBD BDNF or LBD GDNF) to lminin were onduted in vitro. In ddition, the iotivity of LBD BDNF nd LBD GDNF on lminin in vitro ws investigted. LBD BDNF nd LBD GDNF were eh le to speifilly ind to lminin nd mintin their tivity in vitro. Moreover, neurotrophi ftors with NtA retined higher onentrtions nd iotivity levels ompred with those without NtA. The rtio of LBD BDNF nd LBD GDNF tht produed optiml effets ws 4:6. BDNF nd GDNF fused with NtA were effetive in speifilly inding to lminin. As lminin is mjor omponent of the extrellulr mtrix, LBD BDNF nd LBD GDNF my prove useful in the repir of peripherl nerve injuries. Correspondene to: Dr Pin Dong, Deprtment of Otolryngology, Hed nd Nek Surgery, Shnghi Jio Tong University Affilited to Shnghi First People's Hospitl, 100 Hining Rod, Shnghi , P.R. Chin E mil: dongpin64@liyun.om * Contriuted eqully Key words: rin derived neurotrophi ftor, glil ell line derived neurotrophi ftor, immoiliztion, lminin, PC12 ell Introdution The reurrent lryngel nerve (RLN) n e dmged during surgery of the hed nd nek (1). One of the most signifint symptoms of dmge to this nerve is inomplete glotti losure, whih n profoundly derese qulity of life (2). Mediliztion proedures re ommonly rried out to tret unilterl vol fold plsy, solely fulfilling vol fold mediliztion y stti hnges in the lryngel frmework (3). Neurologil impirment n e ompensted for y the regenertion of peripherl nerves. However, experimentl nd linil evidene hs shown tht, prtiulrly fter severe injuries, regenertive ility is usully limited with unstisftory results (1,4 6). Thus, in order to improve reovery following nerve injury, one therpeuti option is to use nturl stimultory regents, suh s neurotrophi ftors. Neurotrophi ftors re le to enhne nerve regenertion, nd therefore my e useful s moleulr therpy. Among ll neurotrophi ftors, rin derived neurotrophi ftor (BDNF) nd glil ell line derived neurotrophi ftor (GDNF) ply ritil roles. BDNF promotes ngiogenesis nd neurl regenertion, s well s modultes lol inflmmtory proesses (7). GDNF hs een shown to oost nerve regenertion fter injury nd exerts survivl promoting effets on motor neurons in vivo nd in vitro (8 11). However, in prtie, lrge proportion of neurotrophi ftors re diffused wy from the site of injury; thus, it is diffiult to retin effetive onentrtions of suh ftors due to their rpid diffusion in extrellulr fluids. One solution to this prolem is repeted injetions of neurotrophi ftors to the injured sites so tht therpeuti onentrtions n e otined; however, this strtegy poses unneessry risks nd osts (12). Thus, the disovery of etter pproh for trgeting neurotrophi ftors to wound sites is impertive (13). One method of overoming the diffusion of neurotrophi ftors is y tking dvntge of their inding ffinity to ertin moleules. In doing so, these ftors n e immoilized t the site of injury. The glyoprotein, lminin, ppers to e n pproprite mteril for trgeting dmged sites euse it is mjor omponent of the extrellulr mtrix (ECM) nd is ioomptile (13). Lminin is minly produed y Shwnn ells nd is widespred in the peripherl nervous

2 WANG et l: IMMOBILIZATION OF NEUROTROPHIC FACTORS TO LAMININ 179 system (PNS) (14,15). Following injury to the PNS, lminin is upregulted nd promotes xonl regenertion (16,17). It hs een reported tht BDNF hs n ffinity for lminin, ut this ffinity is low; more thn hlf of BDNF ound to lminin is quikly relesed within the first dy of injury (13). Thus, the ntive ffinity of BDNF for lminin ppers to e insuffiient for therpeuti purposes. Previous studies hve demonstrted tht the N terminl domin of grin (NtA) hs high ffinity to lminin (18). Mking use of this so lled lminin inding domin (LBD), triprtite fusion protein, whih inluded six histidine purifition tg, LBD, nd the sequene of ntive BDNF or GDNF ws mde in the present study. Thus, the two fusion proteins tht resulted were nmed LBD fused BDNF (LBD BDNF) nd LBD fused GDNF (LBD GDNF). A ntive BDNF nd GDNF without NtA, designted NAT BDNF nd NAT GDNF, respetively, were prepred s ontrols. The overll im of this study ws to utilize lminin s inding trget so tht delivery system ould e onstruted to mintin neurotrophi ftor onentrtions within ertin regions of interest. Mterils nd methods Preprtion of reominnt proteins. LBD BDNF ws prepred s previously desried (13), nd LBD GDNF ws onstruted in n nlogous mnner. GDNF DNA, extrted from the rt ell line PC12 [purhsed from the Cell Bnk of the Chinese Ademy of Sienes (Shnghi, Chin)], ws mplified y polymerse hin retion (PCR) with KOD Plus polymerse kit (Toyoo Co., Ltd., Osk, Jpn). Primer sequenes used were s follows: Forwrd, GGT AGC GGC AGC GGT AGC ACA TGC CCG GAG CGC GCG CTG; reverse, TAC TCG AGT CAG ATA CAT CCA CAC CTT TTAG. Retion onditions for PCR were s follows: Initil denturtion, 95 C for 5 min; 30 yles of denturtion t 95 C for 30 se, nneling t 60 C for 30 se nd extension t 68 C for 30 se; followed y finl extension t 68 C for 5 min. nd inserted into the vetors, pet LBD nd pet 28 (Novgen; EMD Millipore, Sn Diego, CA, USA). Esherihi oli BL21 (DE3) (Biovetor Siene L, In., Beijing, Chin) ws then trnsformed with the vetors, nd proteins were indued with 1 mm isopropyl β D thiogltopyrnoside (IPTG) t 37 C for 5 h. Proteins were umulted in inlusion odies, nd purifition of the soluilized proteins ws performed under denturing onditions y nikel helte hromtogrphy, using imidzole ontining uffer, with HiTrp Chelting High Performne olumn nd n AKTA fst protein liquid hromtogrphy system (Amershm; GE Helthre, Uppsl, Sweden). The purity of the reominnt proteins ws nlyzed y 15% (wt/vol) sodium dodeyl sulfte polyrylmide gel eletrophoresis (SDS PAGE), y whih 20 µg of protein ws seprted nd stined with Coomssie rillint lue solution (Beyotime Institute of Biotehnology, Himen, Chin) for 1 h, then wshed in sline, or trnsferred to PVDF memrne. This ws followed y lok in 5% milk solution, then western lotting with mouse ntiody ginst polyhistidine (t. no ; 1:2,000 dilution; Am, Cmridge, UK). This ws susequently inuted with horserdish peroxidse onjugted got nti mouse ntiody (t. no ; 1:8,000 dilution; Am) for 2 h t room temperture, following whih nds were visulized with enhned hemiluminesene regent (Thermo Fisher Sientifi, In., Wlthm, MA, USA). Purified proteins were then dilyzed in glutthione redox refolding system y hromtogrphi method (19) nd stored t 80 C. In vitro lminin inding ssy. In order to mesure lminin inding pity, modified enzyme linked immunosorent ssy (ELISA) tehnique ws employed in whih lminin (Sigm Aldrih; Merk Millipore, Drmstdt, Germny) ws dded to 96 well pltes (Costr). The pltes were then inuted t 4 C for 24 h, nd ventilted to dry. After wshing the pltes with phosphte uffered sline (PBS; ph 7.3) five times, the pltes were loked with 2.5% (wt/vol) ovine serum lumin (Wuhn Boster Biologil Tehnology, Ltd.- Wuhn, Chin) ontining 0.1% (vol/vol) Tween 20 t 37 C for 2 h. The four reominnt proteins with inresing onentrtions (0 4 µm) were dded to the pltes (100 µl/well) nd inuted t 37 C for 2 h. The pltes were then wshed five times with PBS to remove redundnt proteins. Proteins ound to lminin were tested using mouse ntiody ginst polyhistidine (t. no. H1029; 1:1,000 dilution; Sigm Aldrih) for 1 h t 37 C, followed y got ntiody to mouse IgG (lkline phosphtse onjugted; t. no. P7998; 1:10,000; Sigm Aldrih) for 1 h t 37 C. Bound proteins were deteted using the lkline phosphtse (AP) retion with pr nitrophenylphosphte (pnpp; Sigm Aldrih) in AP uffer (ph 9.6) for 15 min t 37 C; this retion ws stopped using 3 M NOH. Optil density t 405 nm (OD405) ws quntified using n ELISA reder (Moleulr Devies, LLC, Sunnyvle, CA, USA). In vitro lminin relesing ssy. Lminin loded with 1.5 µmol/l NAT BDNF or LBD BDNF or 3 µmol/l NAT GDNF or LBD GDNF ws pled in 48 well plte. Lminin suspension ws indued y the ddition of 500 µl PBS nd inution on roking pltform (37 C, 80 rpm). PBS ws hnged every 24 h, nd t dys 0 7, smples were olleted nd the four proteins retined on the lminin were nlyzed using the ELISA ssy. In vitro iotivity ssy. The iologil tivity of NAT BDNF, LBD BDNF, NAT GDNF nd LBD GDNF ws mesured y neurite outgrowth nd survivl of PC12 ells (20). A density of 3x10 3 ells/well ws seeded in polylysine treted 96 well pltes (Costr) nd ultured in serum free RPMI 1640 medium (Gio; Thermo Fisher Sientifi, In.) t 37 C for 1 h. Inresing onentrtions of NAT BDNF, LBD BDNF, NAT GDNF or LBD GDNF (0 800 ng) were then dded to the pltes. After 24 h of ulture, the perentge of ells with neurites nd the neurite length/ ell dimeter rtio of ells ontining neurites were determined. The numer of surviving ells ws determined using Cell Counting kit (CCK) 8 ssy (Dojindo, Kummoto, Jpn) fter 48 h of ulture. Cells ultured under identil onditions without reominnt proteins served s ontrol. The results were evluted in duplite y two independent reserhers. In vitro iotivity ssy on lminin. Reominnt proteins were seprtely dded to 96 well plte oted with lminin nd inuted t 4 C for 12 h. Pltes were then wshed three

3 180 times to remove ny unound BDNF or GDNF. PC12 ells were seeded t density of 3x10 3 ells/well under the sme onditions s desried in the in vitro iotivity ssy. After 24 h of inution, the ells were oserved using phse ontrst mirosopy (Olympus Corportion, Tokyo, Jpn), nd the perentge of ells with neurites nd length neurite/dimeter of ells ontining neurites were lulted. The numer of surviving ells ws exmined using CCK 8 ssy fter 48 h of ulture. For ontrols, ells were ultured under the sme onditions ut without reominnt proteins. Biotivity of different proportions of LBD BDNF nd LBD GDNF on lminin in vitro. Different proportions of LBD BDNF nd LBD GDNF (1:4, 2:3, 3:2 nd 4:1) were dded to 96 well plte oted with lminin, with the totl protein dded equling 1,000 ng. The reminder of the steps were rried out s previously desried in the iotivity ssy on lminin. Blnk ontrols were mnuftured using the ovementioned onditions, with the exeption tht one of the fusion ftors ws omitted. Cytoskeletl stining. Different proportions of LBD BDNF nd LBD GDNF (1:4, 2:3, 3:2 nd 4:1) were dded to onfol dish oted with lminin. PC12 ells were seeded t density of 3x10 3 under the sme onditions s desried in the iotivity ssy on lminin. The ells were wshed three times with PBS nd fixed with 4% prformldehyde for 2 h t room temperture. Cells were then inuted with 0.5 µg/ml FITC phlloidin (Sigm Aldrih) for 20 min, followed y three wshes with 0.3% Triton X 100. In order to identify ell nulei, 0.1 µg/ml 4',6 dimidino 2 phenylindole (DAPI; Sigm Aldrih) stining ws used. Finlly, fter three wshes in PBS to remove unound DAPI, ells were oserved using onfol fluoresent mirosopy (Olympus Corportion). Sttistil nlysis. All dt were summrized s the men vlue ± stndrd devition. A student's t test ws performed to nlyze pired smples nd one wy nlysis of vrine ws performed to nlyze multiple omprison proedures using Sttistil Pkge for the Soil Sienes (SPSS) version 20.0 softwre (IBM SPSS, Armonk, NY, USA). P<0.05 ws onsidered to indite sttistilly signifint differene. Results Fusion protein strutures. The funtionl modules of NAT BDNF, NAT GDNF, LBD BDNF nd LBD GDNF re shown in Fig. 1. After eing indued y IPTG, the reominnt proteins were expressed, nd identified y western lot nlysis (Fig. 2). LBD BDNF nd LBD GDNF ind to lminin nd exhiit sustined relesed in vitro. The inding of NAT BDNF, LBD BDNF, NAT GDNF nd LBD GDNF to lminin ws mesured in vitro through modified ELISA tehnique. From the results, it ws onluded tht the OD405 of neurotrophi ftors with NtA (the LBD) ws signifintly higher thn tht of neurotrophi ftors without NtA t eh indited point. These findings demonstrte tht the retention of neurotrophi ftors with NtA on lminin ws signifintly greter thn A B Figure 1. Shemti drwing of the engineered (A) NAT BDNF, NAT GDNF, (B) LBD BDNF nd LBD GDNF proteins. His 6, six histidine (n ffinity tg used to purify proteins); LBD, lminin inding domin; BDNF, rin derived neurotrophi ftor (funtionl domin); GDNF, glil ell line derived neurotrophi ftor (funtionl domin); NAT, ntive. C D Figure 2. Sodium dodeyl sulfte polyrylmide gel eletrophoresis of purified proteins. Coomssie lue stining ws used to detet the purifition of (A) LBD BDNF nd (B) LBD GDNF. Western lotting with n ntiody to polyhistidine ws then used to evlute (C) LBD BDNF nd (D) LBD GDNF. LBD, lminin inding domin; BDNF, rin derived neurotrophi ftor; GDNF, glil ell line derived neurotrophi ftor. tht of neurotrophi ftors without NtA (n=6, P<0.05; Fig. 3). Thus, LBD BDNF nd LBD GDNF ould speifilly ind to lminin, nd ftors with NtA possessed stronger lminin inding pity. In the in vitro relese experiment, sustined relese of neurotrophi ftors ws ssessed for 7 dys (Fig. 4). It ws found tht NAT BDNF nd NAT GDNF were quikly relesed on dy 1, wheres LBD BDNF nd LBD GDNF were grdully relesed from dy 1 to dy 7. During the 7 dys, the quntities of LBD BDNF nd LBD GDNF retined on lminin were signifintly greter thn those of NAT BDNF nd NAT GDNF, respetively (n=6, P<0.05). At dy 7, the perentge of NAT BDNF nd NAT GDNF relesed from lminin ws ~87 nd 93%, respetively, wheres the perentge

4 WANG et l: IMMOBILIZATION OF NEUROTROPHIC FACTORS TO LAMININ 181 Figure 3. Lminin inding ssy in vitro. Binding urves of (A) NAT BDNF nd LBD BDNF nd (B) NAT GDNF nd LBD GDNF to lminin mesured y enzyme linked immunosorent ssy. n=6. * P<0.05 vs. NAT ontrol. NAT, ntive; LBD, lminin inding domin; BDNF, rin derived neurotrophi ftor; GDNF, glil ell line derived neurotrophi ftor; OD, optil density. Figure 4. Sustined relese ssy from lminin in vitro. Relese urves of (A) NAT BDNF nd LBD BDN nd (B) NAT GDNF nd LBD GDNF from lminin in vitro. n=6. * P<0.05 vs. NAT ontrol. NAT, ntive; LBD, lminin inding domin; BDNF, rin derived neurotrophi ftor; GDNF, glil ell line derived neurotrophi ftor. Figure 5. Chrteriztion of PC12 ells ws onduted y phse ontrst mirosopy for ells tht were ultured under onditions (A) without reominnt proteins or (B) with LBD BDNF. Blk rrows indite the neurites. Mgnifition, x200. LBD, lminin inding domin; BDNF, rin derived neurotrophi ftor. of LBD BDNF nd LBD GDNF relesed from lminin ws ~57 nd 60%, respetively. These results suggest tht neurotrophi ftors with LBD n e retined on lminin for longer time in vitro thn those without. LBD BDNF nd LBD GDNF mintin higher iotivity on lminin in vitro. PC12 ells were used to test the iotivity of NAT BDNF, LBD BDNF, NAT GDNF nd LBD GDNF. Fig. 5A shows PC12 ells ultured under generl onditions, while Fig. 5B shows ells ultured with LBD BDNF. PC12 ells were oserved fter 48 h inution, nd it ws determined tht the four ftors signifintly promoted neurite outgrowth nd neuronl survivl (Fig. 6), while there ws no signifint differene etween them t eh onentrtion (Fig. 6). This implies tht the fusion of neurotrophi ftors with LBD moiety did not impir the inherent tivity of BDNF or GDNF. The iotivities of BDNF nd GDNF on lminin were then mesured in vitro (Fig. 7). When neurotrophi ftors were inuted in lminin oted wells nd PC12 ells were plted, signifint differene in growth promotion (the perentge of ells with neurites) ompred with ontrols ws oserved (Fig. 7A nd B). Greter numers of living ells nd longer neurites were lso found for the neurotrophi ftors with the LBD NtA (n=6, P<0.05; Fig. 7C F). LBD BDNF nd LBD GDNF mintined higher iotivities, s well s greter onentrtions on lminin thn did the ntive ontrols. These results suggest tht LBD BDNF nd LBD GDNF n trget lminin, nd tht these fusion proteins ould e useful in nerve injury repir. Furthermore, when the rtio of LBD BDNF nd

5 182 C D E F Figure 6. Biotivity omprison of reominnt proteins in vitro. Effet of (A) LBD BDNF, NAT BDNF, (B) LBD GDNF nd NAT GDNF on neurite outgrowth from PC12 ells. Effet of (C) LBD BDNF, NAT BDNF, (D) LBD GDNF nd NAT GDNF on the rtio of neurite length/ell dimeter in PC12 ells. Effet of (E) LBD BDNF, NAT BDNF, (F) LBD GDNF nd NAT GDNF on PC12 ell survivl s determined y Cell Counting kit 8 ssy. Dt re presented s men ± stndrd devition (n=6). NAT, ntive; LBD, lminin inding domin; BDNF, rin derived neurotrophi ftor; GDNF, glil ell line derived neurotrophi ftor; OD, optil density. LBD GDNF ws 4:6, the ility to promote neurite growth ws superior to tht of the other rtios exmined (Fig. 8). Cytoskeletl stining. As shown in Fig. 9, the ytoskeleton, nuleus nd neurites of the PC12 ells re lerly presented. Although the entire ell skeleton nnot e visulized, the fetured imges were hosen for their ler presenttions of the neurites. After omining LBD BDNF nd LBD GDNF, greter effet ws oserved thn when they were pplied seprtely. Through these results, it my e onluded tht when the rtio of mixing of LBD BDNF nd LBD GDNF ws 4:6, their ility to promote growth ws superior to tht t other rtios, demonstrting synergeti effet (Fig. 8). Disussion Peripherl nerve injury often uses loss of funtion, nd the urrent gold stndrd for repiring suh dmge is utologous nerve grfting (21). However, the linil pplition of utologous nerve grfting is hmpered due to limited donor sites, extr inisions required, nd possile loss of funtion t the donor site (21). One potentil therpeuti strtegy tht ould ddress these issues is the pplition of nturl stimultory ftors, suh s neurotrophi ftors, to the site of injury. Neurotrophi ftors re produed y orgnisms to promote neurl ell survivl, growth nd differentition. Neurotrophi ftors re not only le to redue nerve degenertion nd prevent the progression of disese, ut re lso le to stimulte xonl growth nd promote funtionl regenertion (22). Reserhers hve studied severl fusion neurotrophi ftors, inluding ntive humn nerve growth ftor (NGF) β fused with ollgen inding domin (CBD) (23 25), fironetin fused with CBD (26), BDNF fused with CBD (7,27 32), NGF β fused with LBD (20), iliry neurotrophi ftor fused with LBD (33), nd BDNF fused with LBD (13); however, to the est of our knowledge, to dte there hve een no studies investigting n LBD GDNF fusion protein. BDNF nd GDNF ply importnt roles in the repir of nerve injury. However, it is diffiult to retin them t injury sites euse they esily diffuse. In order to mintin tive onentrtions, previous studies hve employed multiple injetions, denovirl vetors overexpressing BDNF or GDNF, or fusion proteins suh s CBD BDNF nd LBD BDNF (3,7,13,34,35). However, the risk of surgery, immunologil rejetion, nd

6 WANG et l: IMMOBILIZATION OF NEUROTROPHIC FACTORS TO LAMININ 183 C D E F Figure 7. Biotivity omprison of reominnt proteins on lminin in vitro. Effet of (A) LBD BDNF, NAT BDNF, (B) LBD GDNF nd NAT GDNF on neurite outgrowth from PC12 ells. Effet of (C) LBD BDNF, NAT BDNF, (D) LBD GDNF nd NAT GDNF on the rtio of neurite length/ell dimeter in PC12 ells. Effet of (E) LBD BDNF, NAT BDNF, (F) LBD GDNF nd NAT GDNF on PC12 ell survivl s determined y Cell Counting kit 8 ssy. Dt re presented s men ± stndrd devition (n=6). * P<0.05 the NAT ontrol. NAT, ntive; LBD, lminin inding domin; BDNF, rin derived neurotrophi ftor; GDNF, glil ell line derived neurotrophi ftor; OD, optil density. C Figure 8. Biotivity omprison of different porportions of reominnt proteins on lminin in vitro. Effet of different porportions of reominnt proteins on (A) neurite outgrowth nd (B) the the rtio of neurite length/ell dimeter in PC12 ells. (C) Effet of different proportions of reominnt proteins on ell survivl in PC12 ells y Cell Counting kit 8 ssy. Blnk ontrols (rtio 0 or 10) indites ells were ultured under similr onditions with the exeption tht LBD BDNF or LBD GDNF ws omitted. Dt re presented s men ± stndrd devition (n=6). * P<0.05 vs. NAT ontrol. The sterisk within squre indites the optimum rtio of mixing to promote growth. NAT, ntive; LBD, lminin inding domin; BDNF, rin derived neurotrophi ftor; GDNF, glil ell line derived neurotrophi ftor; OD, optil density. osts with these methods would ll e inresed. Moreover, the presene of redundnt neurotrophi ftors ould use dverse effets (7). Lminin, ritil omponent of the ECM in the PNS, hs een shown to ffet neuronl ehvior, inluding migrtion, neurite outgrowth, prolifertion, nd entrl synpti differentition (20,36). A previous study hs shown tht LBD BDNF n support mrked neuroprotetive funtion following middle ererl rtery olusion (13). Therefore, in n ttempt to retin neurotrophi ftors t the site of injury nd enhne nerve regenertion, lminin ws hosen s trget for BDNF nd GDNF in the present study. Whether

7 184 A B C D E F Figure 9. Imges of PC12 ell (A) ytoskeletons; (A) nuleim, or (A) overlys, viewed y onfol fluoresent mirosopy following tretment with different proportions of LBD BDNF nd LBD GDNF. (A) Only LBD BDNF, LBD GDNF nd LBD BDNF in (B) 1:4 rtio, (C) 2:3 rtio, (D) 3:2 rtio nd (E) 4:1 rtio, nd (F) only LBD GDNF. NAT, ntive; LBD, lminin inding domin; BDNF, rin derived neurotrophi ftor; GDNF, glil ell line derived neurotrophi ftor. LBD BDNF nd LBD GDNF hd synergisti effets ws lso evluted. Agrin is synpse orgnizer tht promotes etylholine reeptor lustering t the neuromusulr juntion. Reserh hs shown tht NtA hs high ffinity with the oiled oil domin of lminin (18). Thus, in order to trget BDNF nd GDNF to lminin, NtA ws fused to the two neurotrophi ftors to rete LBD BDNF nd LBD GDNF in the present study. As expeted, ompred with NAT BDNF nd NAT GDNF, LBD BDNF nd LBD GDNF presented omprle lminin inding

8 WANG et l: IMMOBILIZATION OF NEUROTROPHIC FACTORS TO LAMININ 185 ility nd demonstrted neuroprotetive tivities. In the lminin inding ssy of LBD BDNF nd LBD GDNF, neurotrophi ftors with NtA showed stronger inding ilities to lminin ompred with those without NtA. At the sme onentrtion, neurotrophi ftors with NtA ould e trgeted to lminin, nd effetively voided eing wshed wy or extensively diluted y extrellulr fluids. Thus, LBD BDNF or LBD GDNF served s trgeted proteins for mintining effetive onentrtions of neurotrophi ftors. Next, PC12 ells were used for evluting the iotivities of LBD BDNF nd LBD GDNF through neurite outgrowth nd the CCK 8 ssy. The results showed tht LBD BDNF nd LBD GDNF mintined the iotivities of BDNF nd GDNF. Additionlly, neurotrophi ftors with NtA retined higher onentrtions nd iotivities ompred with those without NtA. It my e onluded tht this ws due to differene in lminin inding ffinity etween these proteins. Further, these findings showed tht NtA enled BDNF nd GDNF trget lminin. Finlly, LBD BDNF nd LBD GDNF were mixed together in vrying rtios to detet whih rtio of mixing ws the most onduive for the growth of PC12 ells. Optiml results were oserved when LBD BDNF nd LBD GDNF were mixed in rtio of 4:6. Moreover, it ws found tht mixture of LBD BDNF nd LBD GDNF promoted the growth of PC12 ells to greter extent thn the use of either LBD BDNF or LBD GDNF lone t the orresponding onentrtion. The present in vitro experiments lerly showed tht these fusion proteins hd ertin dvntges over ntive ones. Thus, the ove findings provide evidene for the use of fusion proteins to immoilize moleule, suh s BDNF or GDNF, on lminin oted surfe. There re mny methods for the pplition of exogenous neurotrophi ftors; however, it ppers tht the most effetive pprohes for mintining the survivl of motor neurons nd promoting their xonl regenertion is vi routes tht ensure suffiient onentrtions nd the ontinuous funtion of neurotrophi ftors. The disdvntges of lolly injeting neurotrophi ftors inlude poor outome, short lived enefits, nd side effets (30). If the dministrtion of suh ftors ould e ontrolled effetively nd urtely t the injured re, they ould e more sfe nd effetive. In ddition, there re few studies tht hve reported on the pplition of neurotrophi ftors to regenertion of the RLN. Further si reserh nd dvnements in the linil development of RLN repir tehnology should llow for etter understnding of this pproh. Moreover, linil nd si reserh of the dministrtion of neurotrophi ftors vi this tehnology is likely to promote the tretment of RLN injury nd other diseses of the nervous system. 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