Two Monoclonal Antibodies Recognising aa 634-668 and aa 1026-1055 of NogoA Enhance Axon Extension and Branching in Cultured Neurons

In a previous study, we generated two monoclonal antibodies (mAbs) in mice, aNogoA-N and aNogo-66 mAb, which were raised against recombinant N-terminal fragments of rat NogoA and Nogo-66, respectively. When compared with the commercial rabbit anti-rat NogoA polyclonal antibody (pAb), which can specifically recognise NogoA, the two mAbs were also specific for the NogoA antigen in immunofluorescence histochemical (IHC) staining and Western blot (WB) analysis. Serial truncations of NogoA covering the N-terminal region of NogoA (aa 570–691) and Nogo-66 (aa 1026–1091) were expressed in E. coli. The epitopes recognised by aNogoA-N and aNogo-66 are located in the aa 634–668 and aa 1026–1055 regions of NogoA, respectively. Both mAbs remarkably enhanced the axon growth and branching of cultured hippocampal neurons in vitro. These results suggest that the antibodies that bind to aa 634–668 and aa 1026–1055 of NogoA may have stimulatory effects on axon growth and branching. Additionally, the two mAbs that we generated are specific for NogoA and significantly block NogoA function. In conclusion, two sites in NogoA located within aa 634–668 and aa 1026–1055 are recognised by our two antibodies and are novel and potentially promising targets for repair after central nervous system (CNS) injury.


Introduction
After injury, the central nervous system (CNS) of adult mammals is limited in its ability to recover because of the inability of damaged axons to reconnect and regain their physiological structure and function [1]. Factors that influence axon regeneration include neural cell-autonomous activity, glial scarring, local inflammation, and inhibition factors [2,3]. In the past two decades, several CNS myelin-derived axon growth inhibitory factors have been found, including NogoA, myelin-associated glycoprotein (MAG), and oligodendrocyte myelin glycoprotein (OMgp) [4,5,6]. These proteins have been the subject of great research interest and are highly clinical relevant. NogoA plays an important role in recovery from spinal cord injury, oligodendrocyte differentiation and myelination [7,8,9], and the development of the CNS [10,11]. A recent study reported that a region of NogoA (aa 290-562) attenuates cerebral ischaemia by inhibiting NADPH oxidasemediated oxidative damage and neuronal apoptosis [12], indicating that the functional diversity of different fragments of NogoA must be explored.
The function of NogoA has been studied extensively using anti-NogoA antibodies [13,14]. In a previous study, we developed two anti-NogoA monoclonal antibodies, aNogoA-N and aNogo66 mAb, which were generated in mice using recombinant aa 570-691 and aa 1026-1091 fragments, respectively, from NogoA [15]. In the present study, we analysed the specificity and affinity of the two mAbs to the NogoA molecule. We also detected the different epitopes in NogoA that could be recognised by the two mAbs. Using in vitro experiments, we found that these mAbs against NogoA enhanced axon growth and branch formation.

Western blot and IHC staining
The protein extract from the spinal cord tissues of Sprague-Dawley rats was separated by 10% sodium dodecyl sulphatepolyacrylamide gel electrophoresis (SDS-PAGE) and transferred onto Hybond-P PVDF membranes (Amersham Biosciences) using the Trans-Blot SD Semi-Dry Transfer cell (Bio-Rad) following the manufacturer's instructions. One transferred membrane was blocked with 3% skim milk and 3% bovine serum albumin (BSA) in PBS containing 0.1% Tween-20 for 2 h and incubated with the commercial anti-NogoA pAb (1:500, 1:5000, 1:20000), which was used as positive control, and the other two transferred membranes were incubated with aNogo66 mAb and aNogoA-N mAb (1:500, 1:5000, 1:20000) (1 mg/mL stock concentration) at 4uC overnight. The membranes were washed three times with washing buffer (PBS, 0.05% Tween-20) and then incubated with HRP-conjugated goat anti-mouse IgG or HRP-conjugated goat anti-rabbit IgG (1:5000 dilution in blocking buffer) (Rockland) for 1 h at room temperature. The membranes were washed three times with washing buffer before antibody binding was visualised using enhanced chemiluminescence reagents (Lumiglo TM ; Cell Signaling).
The method used to test the binding of antibodies to the targeted Nogo-A region was as follows: The NogoA FC-(aa 1026-1090) or NogoA FC-(aa 544-725) protein was separated by 10% sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) and transferred onto Hybond-P PVDF. Blots were probed with aNogo66 mAb or aNogoA-N mAb (1:500) at 4uC overnight and incubated with HRP-conjugated goat anti-mouse IgG (1:5000) (Rockland) for 1 h at room temperature. The membranes was visualised using an enhanced chemiluminescence reagent (Lumiglo TM ; Cell Signaling).
To detect growth-associated protein 43 (Gap-43) expression, the cultured primary neurons were harvested on the fifth and seventh days, and the total protein concentration of the cells was analysed using a BCA kit (Sigma, CA, USA). Blots were probed with a mouse monoclonal antibody against Gap-43 (1:500, Santa Cruz, CA, USA) and b-actin (1:2000; Anbo, USA). Each blot was incubated for 2 h at room temperature. Then, the blots were incubated with HRP-conjugated goat anti-mouse IgG (1:5000 dilution in blocking buffer) (Rockland) for 1 h at room temperature. The membranes was visualised using an enhanced chemiluminescence reagent (Lumiglo TM ; Cell Signaling).
For IHC, adult rats were anesthetised by an intraperitoneal injection of an overdose of sodium phenobarbital (100 mg/kg) and were then perfused intracardially with warm saline followed by 4% paraformaldehyde (PFA) (pH 7.4). After perfusion, a 15-mm-length thoracolumbar segment of the spinal cord was removed and put into 25% sucrose in 0.1 M phosphate buffer for 36 h at 4uC. Serial coronal sections of a 12 mm thickness were prepared using a freezing microtome (Leica, CA1900, Germany). The sections were post-fixed in 4% PFA for 1 h at room temperature. Subsequently, the sections were rinsed with 0.01 M phosphate-buffered saline (PBS) and then blocked with 1% BSA (Sigma, USA) in PBS containing 0.3% Triton X-100 for 1 h at room temperature. The sections were divided into six groups for the different primary antibodies: I, aNogo66 mAb and Anti-NogoA pAb; II, aNogoA-N mAb and Anti-NogoA pAb; III, aNogo66 mAb and anti-MBP mAb; IV, aNogoA-N mAb and anti-MBP mAb; V, aNogo66 mAb and anti-GFAP mAb; VI, aNogoA-N mAb and anti-GFAP mAb. All sections were incubated in primary antibody at 4uC for 24 h. After washing with PBS three times, the secondary antibodies were incubated in a dark environment at room temperature for 2 h. The stained sections were then washed with PBS three times and mounted with glycerol. The sections were observed under an Olympus BX-51 microscope. After sequencing, all plasmids containing the truncated fragments were transformed into BL21 (DE3) E. coli for expression. The recombinants were GST-DNogoA-Na, 43 kDa; GST-DNo-goA-Nb, 38 kDa; GST-DNogoA-Nc, 33 kDa; GST-DNogoA-Nd, 30 kDa; GST-DNogo66a, 33 kDa; GST-DNogo66b, 30 kDa. The GST protein is 26 kDa and was expressed from the empty pGEX-4T-1 vector. In this study, the expression of recombinant proteins was under the control of the Tac promoter and was induced by isopropyl-b-D-thiogalactopyranoside (IPTG; Sigma-Aldrich Co., St. Louis, MO). After 2 h of shaking, a final concentration of 0.5 mM IPTG was added into the Luria-broth (LB) medium with ampicillin (100 mg/L), and the medium was further agitated for 3 h [16]. Subsequently, the bacterial cells were pelleted at 12000 g for 6 min, buffer was added, and the cells were boiled for 10 min. SDS-PAGE was performed to analyse the molecular size of the recombinant proteins using Coomassie blue staining and WB analysis. WB was performed briefly as follows. The antibodies were diluted as follows: rabbit anti-GST antibody was diluted 1:30000 in blocking solution and incubated with the blots for 2 h at room temperature; the secondary antibodies were (HRP)-conjugated goat anti-rabbit IgG (1:5000) and were incubated with the blots for 1 h at room temperature. Antibody binding was visualised using an enhanced chemiluminescence reagent (Lumiglo TM; Cell Signaling).

Expression and purification of recombinant proteins
The recombinant proteins were purified by affinity chromatography using a pre-charged Ni-NTA Sepharose column (Qiagen Inc., Valencia, CA). The dialysis purity was verified by SDS-PAGE, and the protein concentration was determined by the bicinchoninic acid (BCA) protein quantitation method (Nanjing Jiancheng, China) (Data not shown). Samples were stored at 220uC until use.

Axon outgrowth and branch formation
Glass coverslips (1 cm) were coated with poly-L-lysine (5 mg/ mL), washed three times, and subsequently coated with NogoA FC (aa 1026-1090) or NogoA FC (aa 544-725) (100 pmol, diluted in PBS) for 2 h at 37uC. Unbound NogoA was removed by three washes with PBS. To evaluate the blocking function of the mAbs, glass coverslips were coated with two mAbs (30 mg/ml) for 1 h at 37uC.
Primary hippocampal neurons cultures were prepared as previously described [17]. Briefly, the hippocampal neurons were acquired from SD rat embryos (E18.5). The pups were anesthetised, and 75% ethanol was sprayed on the animals for 5 min. The neurons were isolated and washed with D-Hank's solution three times under sterile conditions. Cells were seeded at a density of 1610 6 cells/cm 2 onto plates and maintained in a humidified incubator (Forma Scientific CO 2 3110, Thermo Electron Corporation, USA) at 5% CO 2 and 37uC. The neurons were cultured in Neurobasal (Gibco, Invitrogen Corp., CA, USA) supplemented with 2% B27 (Gibco, Invitrogen Corp., CA, USA), 1% glutamine (Sigma-Aldrich Corp., St. Louis, MO, USA), and 1% penicillin/streptomycin (Sigma-Aldrich Corp, USA). Half of the medium was changed twice a week. The purity of neurons was determined by immunocytochemistry for bIII-tubulin, and the analysis indicated that 95% of the cells in the cultures were bIIItubulin (1:250; Anbo, USA) positive (data not shown).
To observe axon outgrowth, cells were used for immunostaining on the seventh day after culture. The cells were washed after fixation in 4% PFA and then stained with anti-Map2 (1:1000) and anti-Tau (1:1000). Measurement of axon length was performed as follows. Five randomly chosen fields of view from the coverslips were photographed using a phase-contrast Olympus IMT2 microscope and an F-View camera at 206 magnification. The axon length per Tau-stained neuron was then measured from these photographs. Statistical significance was assessed using Student's t test. The measurements of the total number of Taupositive neurons from three independent experiments were analysed by the t-test.
To observe branch formation, the number of axon branch points of per neuron and the distance at which the axon sent out its first branches from the cell body were measured using a phasecontrast Olympus IMT2 microscope and an F-View camera at 406 after examining five randomly chosen fields of view from the coverslips. Statistical significance was assessed using Student's t test. The measurements from three independent experiments were analysed by the t test.
Statistical analysis SPSS 13.0 software for Windows (SPSS Inc., Chicago, IL) was used for the statistical analyses. All the data are presented as the means 6 SEM. Differences between the groups were assessed by one-way ANOVA followed by the LSD-t test. P values ,0.05 were considered to be significant.

Results
The two mAbs we generated specifically recognised NogoA protein The specificity and the affinity of the two mAbs were tested by WB (Fig. 1A). The two mAbs and the commercial rabbit anti-NogoA polyclonal antibody bound to NogoA from spinal cord tissue, with corresponding bands at 200 kDa. Additionally, aNogo66 mAb and aNogoA-N mAb, at different concentrations, strongly bound the NogoA molecule at 200 kDa, indicating that the two mAbs specifically recognise NogoA and have a good affinity for NogoA.
NogoA localises to the membrane surface, the cytoplasm, and processes in oligodendrocytes. First, IHC staining was used to determine the reactivity and specificity of the mAbs in spinal cord tissue from rats. The aNogo-N mAb and aNogo-66 mAbs were double-labelled with commercial rabbit anti-NogoA polyclonal antibody (pAb) in spinal cord sections (Fig. 1B). Furthermore, the two mAbs colocalised with MBP, which is a positive marker for oligodendrocyte neurites and myelin (Fig. 1C). However, the two mAbs did not double-stain with GFAP (Fig. 1D). These results suggest that the two mAbs specifically recognise NogoA.
The epitopes recognised by the aNogo-N and aNogo-66 mAbs are located within aa 634-668 and aa 1026-1055 of NogoA, respectively To identify the location of the epitopes in NogoA that are recognised by the aNogo-N and aNogo-66 mAbs, six truncations of NogoA were produced as recombinant GST-fused peptides ( Fig. 2A). All the recombinants displayed their predicted molecular weights after SDS-PAGE and Coomassie blue staining (Fig. 2B). Subsequently, we used an anti-GST antibody to detect these recombinant proteins by WB. Recombinant proteins in all lanes were recognised specifically by the anti-GST antibody except for lane 8, where the protein was not expressed without IPTG induction (Fig. 2C). Next, the GST-tagged fusion proteins were purified by Ni-NTA agarose affinity chromatography. After the bound proteins were eluted, the purity of the final products in the portion of the eluate was approximately 95% (data not shown).
To detect the location of the epitopes in NogoA that are recognised by aNogo-N mAb and aNogo-66 mAb, we screened the recombinants by WB assay. The aNogoA-N mAb recognised the GST-DNogoA-Na, GST-DNogoA-Nb, and GST-DNogoA-Nc peptides but not the GST-DNogoA-Nd peptide (Fig. 2D). This result suggested that the epitope recognised by aNogoA-N mAb is located between aa 634 and 668 of NogoA (Table S1). Using the same method, the aNogo66 mAb only bound the GST-DNogo66a peptide but did not recognise the GST-DNogo66b peptide (Fig. 2E). This result suggested that the epitope recognised by  aNogoA-N mAb is located between aa 1026 and 1055 of NogoA (Table S1).

Both mAbs prevented the inhibition of NogoA fragments on axonal extension and branching in vitro
Hippocampal neurons contain a long axon under normal culture conditions in vitro (Fig. 3A). The axon extension of cultured hippocampal neurons was significantly inhibited by NogoA FC-(aa 1026-1090) or NogoA-FC (aa 544-725) treatment in vitro (P, 0.01) (Fig. 3B, D, E). Compared with the control group, axon extension was almost completely rescued by adding aNogoA-N mAb to the NogoA FC-(aa 544-725) treated group or by adding aNogo66 mAb to the NogoA FC-(aa 1026-1090) treated group (Fig. 3C, D, E). We next examined the function of the mAbs on axonal branching in cultured neurons in vitro. The number of branching points in the mAb groups was greater than either of the NogoA FC groups (P,0.05) (Fig. 4A, B, C). The axons of cells in the mAb groups sent out their first branches farther away from the cell body than did axons from cells in either of the NogoA FC groups (P,0.01) (Fig. 4 A, C, D). These results showed that the two mAbs we generated can block NogoA fragments that inhibit axon extension and branching in vitro.
The axon extensions were almost completely rescued by adding the aNogoA-N mAb to the NogoA FC-(aa 544-725) or by adding the aNogo66 mAb to the NogoA FC-(aa 1026-1090) (Fig. 5 C/ G). However, aNogoA-N could not rescue axon outgrowth on The neurons treated with the two mAbs upregulated GAP-43 in vitro GAP-43 plays a critical role in axonal extension and branching [1]. We next assayed the expression level of GAP-43 in cultured neurons after mAb treatment in vitro (Fig. 6). The level of GAP-43 was significantly higher in both the aNogo-66 mAb and aNogoA-N mAb treatment groups than the NogoA FC (aa 1026-1090) and NogoA FC (aa 544-725) groups. These results implied that NogoA may inhibit axonal extension and branching via the downregulation of GAP-43.

Discussion
Rat NogoA is a member of the reticulon family of transmembrane proteins. The full-length rat NogoA is 1163 aa in length and contains a 989 aa N-terminus, a 21 aa transmembrane segment, a 94 aa connecting ''loop'', a second 21 aa transmembrane segment, and a 38 aa C-terminus [18]. The potent inhibitory activities of the different NogoA regions have been studied previously. Three areas are of particular interest. One such area is Nogo-66 (aa 1026-1091) in the C-terminal region of NogoA, which is reported to bind to the GPI-linked Nogo receptor/p75 complex on axons and induce growth cone collapse [19,20]. Two other regions in the Nterminus have also been discovered to have bioactivity. Amino acids 59-172 are reported to block fibroblast spreading, whereas Nogo-D20 (aa 544-725) exerts strong inhibitory effects on growing neurites and growth cones in vitro and, unlike Nogo-66, also on the migration of non-neuronal cells such as fibroblasts [21,22,23].
Furthermore, an anti-serum (AS 472) against aa 623-640 in the NogoA-specific region neutralises the inhibitory activity of CNS myelin in vitro and induces the sprouting of adult rat Purkinje axons in vivo [21]. Using function-blocking NogoA-specific antibodies to block NogoA or a soluble Nogo-66-binding fusion protein comprising the domains of NgR1, using antagonistic peptides, or blocking Rho-A and its downstream target ROCK can improve regeneration [1].
In our previous study, we developed two different monoclonal antibodies, aNogo66 and aNogoA-N mAbs, which were produced against aa 570-691 and aa 1026-1091 of the rat NogoA protein [15]. However, there were many basic research and clinical application questions to be explored. For example, the epitopes of the mAbs needed to be identified, and the blocking function of the mAbs needed to be elucidated.
In the present study, we found that the two mAbs spefocifically recognise NogoA in tissues. The aNogo66 mAb recognises an epitope within aa 1026-1055. Interestingly, another region of Nogo66 (aa 1055-1099 of NogoA), on the surface of oligodendrocytes, can be recognised by the antibody AS 922, which can block the neurite growth inhibitory activity of NogoA [21]. Therefore, aa 1026-1055 may be a new functional region in NogoA, and the aNogo66 mAb may have valuable applications in the future. The aNogoA-N mAb recognises an epitope within aa 634-668, which is different from the synthetic peptide corresponding to the rat sequence (aa 623-640) for mAb 11C7, which enhances axon growth and fibroblast spreading [23]. Other reports have shown that amino-NogoA antagonises reactive oxygen species generation and protects immature primary cortical neurons from oxidative toxicity [24]. These findings suggested that aa 634-668 may be another new functional region of NogoA.
NogoA is highly expressed in outgrowing neurons in vivo [25,26], including in growth cones. In the CNS, adult mice lacking NogoA showed an upregulation of cytoskeletal and growth-related mRNAs and proteins in the spinal cord and cortex [27]. Furthermore, the addition of function-blocking NogoA-specific antibodies induces both the upregulation of growth-specific proteins and pronounced neurite sprouting in hippocampal neurons [28]. Notably, during these processes, GAP-43 plays an important role coincident with myelin formation [29,30,31,32]. A remarkable congruence has been found in the effects of different blocking agents for NogoA signalling, including antibodies, receptor bodies, and small molecule blockers, in a number of regeneration and plasticity paradigms [1,33,34]. An antibody that blocks NogoA function has reached the clinical trial stage as a novel treatment for spinal cord injury [14,35,36].
NogoA exerts repulsive and neurite growth-inhibitory functions in the CNS of developing and adult animals and can be found in the innermost membrane and in the outer myelin membrane in oligodendrocytes [25,37]. Additionally, NogoA is highly expressed in outgrowing neurons in vivo, including in growth cones and at synapses [37]. Nogo receptor 1 (NgR1) is one part of the functional Nogo receptor complex with proteins such as LINGO1 and the presumed signal transducers p75 and Troy [38]. In addition, NgR1 is expressed on the nerve cell body, in growth cones, and at synapses [19]. In vivo, the acute blockade of NgR1 enhanced sprouting, regeneration, and plastic rearrangements of fibre connections after CNS injury in adult rats [39,40]. Thus, a NogoA antibody can inhibit NogoA-NgR binding. Treatments targeting Nogo signalling resulted in the most consistent and extensive structural and functional recoveries after spinal cord or stroke lesions [40,]41,42]. A clinical study in patients with acute injuries in the spinal cord with a human NogoA antibody is currently underway. Therefore, our research may have important applications for basic to clinical studies of spinal cord or stroke lesions.
In summary, the epitope sequences recognised by the aNogo66 mAb and aNogoA-N mAb were determined, and the specificity and affinity of the mAbs to NogoA were demonstrated. Furthermore, we demonstrated that both mAbs reverse the inhibitory effect of NogoA protein on axon growth and branch formation in vitro. Moreover, these findings suggest that the epitope sequences may constitute new functional regions of NogoA that can regulate growth cone collapse and neuronal plasticity. The mAbs may benefit studies of the function of NogoA, particularly studies of target and short-peptide drugs for the treatment of CNS injury.

Supporting Information
Table S1 Amino acid sequences of peptide of Rat NogoA recognised by aNogo66 mAb or aNogoA-N mAb, respectively. (DOCX)