Improvement of Sciatic Nerve Regeneration Using Laminin-Binding Human NGF-β

Citation: Sun W, Sun C, Zhao H, Lin H, Han Q, et al. ( Improvement of Sciatic Nerve Regeneration Using Laminin-Binding Human NGF-b Wenjie Sun Changkai Sun Hui Zhao Hang Lin Qianqian Han Jingyu Wang Hui Ma Bing Chen Zhifeng Xiao Jianwu Dai Xiao-Jiang Li, Emory University School of Medicine, United States of America Background: Sciatic nerve injuries often cause partial or total loss of motor, sensory and autonomic functions due to the axon discontinuity, degeneration, and eventual death which finally result in substantial functional loss and decreased quality of life. Nerve growth factor (NGF) plays a critical role in peripheral nerve regeneration. However, the lack of efficient NGF delivery approach limits its clinical applications. We reported here by fusing with the N-terminal domain of agrin (NtA), NGF-b could target to nerve cells and improve nerve regeneration. Methods: Laminin-binding assay and sustained release assay of NGF-b fused with NtA (LBD-NGF) from laminin in vitro were carried out. The bioactivity of LBD-NGF on laminin in vitro was also measured. Using the rat sciatic nerve crush injury model, the nerve repair and functional restoration by utilizing LBD-NGF were tested. Findings: LBD-NGF could specifically bind to laminin and maintain NGF activity both in vitro and in vivo. In the rat sciatic nerve crush injury model, we found that LBD-NGF could be retained and concentrated at the nerve injury sites to promote nerve repair and enhance functional restoration following nerve damages. Conclusion: Fused with NtA, NGF-b could bind to laminin specifically. Since laminin is the major component of nerve extracellular matrix, laminin binding NGF could target to nerve cells and improve the repair of peripheral nerve injuries. - Funding: This work was supported by grants from National Natural Science Foundation of China (30688002, 30800564), Chinese Academy of Sciences (KSCX2YW-R-133), and the Ministry of Science and Technology of China (2006CB943601). The authors also gratefully acknowledge the support of K.C.Wang Education Foundation. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: The authors have declared that no competing interests exist. Sciatic nerve injuries are often caused by injections, gunshot wounds, lacerations, contusions, compressions, and iatrogenic causes [1,2]. Injuries to sciatic nerves cause partial or total loss of motor, sensory and autonomic functions due to the axon discontinuity, degeneration, and eventual death which finally result in substantial functional loss and decreased quality of life [3,4]. Functional deficits caused by nerve injuries can be compensated by regeneration of peripheral nerves. However, clinical and experimental evidences show that the regeneration is usually far more difficult and the results are far from satisfactory, especially after severe injuries [3,5]. Numerous therapeutic interventions, mostly pharmacotherapeutic, have been tested to enhance functional recovery after sciatic nerve injuries. The identification of neurotrophic factors offers molecular therapy as a potential approach to enhance nerve regeneration. Among the neurotrophic factors, nerve growth factor (NGF) plays a critical role. NGF promotes proliferation and differentiation of neurons, and also modulates the repair of injured nerves [6,7]. It has been reported that NGF was upregulated in anticipation of the arrival of a regenerating sprout during the peripheral nerve regeneration [8]. The administration of recombinant NGF protein into injured nerves has been shown to promote nerve repair and enhance functional restoration following nerve damages [9]. However NGF simply given in solution is difficult to be retained at the injury sites because of its rapid diffusion in body fluids. Therefore, it requires periodic injection of NGF which is impractical and expensive, and excessive doses may also evoke undesirable side effects [1012]. To solve these problems, many groups are working on developing NGF delivery to the nervous system via drug delivery systems [13] or transplantation of cells with/without encapsulation [14]. These systems should be improved with regard to release control, dosing, efficacy and safety. Laminin is the ubiquitous component of the tight network of glycoproteins, collagen IV and proteoglycans in basement membranes [15]. Laminin is mainly produced by Schwann cells and widely dispersed in the peripheral nervous system (PNS) [16,17]. After peripheral nerve injuries, laminin is significantly upregulated at the injury sites by Schwann cells and may foster axonal regeneration [18,19]. Thus, laminin could be a suitable target for the delivery of exogenous NGF to repair PNS injuries. Agrin is a key organizer of postsynaptic differentiation at the neuromuscular junction. The binding of agrin to laminin is required for its localization to synaptic basal lamina and other basement membranes [20]. Previous studies have demonstrated that the high-affinity interaction with the coiled-coil domain of laminin was mediated by N-terminal domain of agrin (NtA) [21]. Taking advantage of this laminin-binding domain of NtA, we produced a tripartite fusion protein to obtain laminin binding NGF-b which contains (i) the primary sequence of the mature NGF-b, (ii) NtA as a laminin-binding domain, and (iii) a 66histidine (His) purification tag. This fusion protein was named as laminin binding domain fused NGF-b (LBD-NGF). We also produced a native NGF-b without NtA (NAT-NGF). We demonstrated that the LBD-NGF could specifically bind to laminin and maintain NGF activity both in vitro and in vivo. In the rat sciatic nerve crush injury model, LBD-NGF could target to the native nerve extracellular matrix component laminin, and could be retained and concentrated at the nerve injury sites to improve peripheral nerve regeneration. LBD-NGF could bind to laminin and be sustained released in vitro We measured the in vitro laminin-binding activities of NATNGF and LBD-NGF by ELISA. From the results, we found at each indicated point the OD405 of LBD-NGF was significantly higher than that of NAT-NGF, indicating that the retained LBDNGF on laminin was more than that of NAT-NGF (n = 6, P,0.01) (Figure 1A). Using the binding curve, we calculated the dissociation constant Kd values of NAT-NGF and LBD-NGF to laminin by Scatchard analysis. The slope of the resulting straight line equals 21/Kd (Figure 1B). The Kd value for the binding of NAT-NGF and LBDNGF was measured as 6.2561024 M and 7.2561026 M respectively. The lower Kd value of LBD-NGF to laminin indicated that LBD-NGF bound to laminin specifically. Using ELISA assay, we found the membrane prepared from pig amnion was rich in laminin content (Figure 1C). In the in vitro release experiment, sustained release of NAT-NGF and LBDNGF was followed up to 8 days (Figure 1D). We found NAT-NGF was quickly released at the (...truncated)