title: Oncomodulin/truncated protamine-mediated Nogo-66 receptor small interference RNA delivery promotes axon regeneration in retinal ganglion cells.
abstract: The optic nerve often suffers regenerative failure after injury, leading to serious visual impairment such as glaucoma. The main inhibitory factors, including Nogo-A, oligodendrocyte myelin glycoprotein, and myelin-associated glycoprotein, exert their inhibitory effects on axonal growth through the same receptor, the Nogo-66 receptor (NgR). Oncomodulin (OM), a calcium-binding protein with a molecular weight of an ∼12 kDa, which is secreted from activated macrophages, has been demonstrated to have high and specific affinity for retinal ganglion cells (RGC) and promote greater axonal regeneration than other known polypeptide growth factors. Protamine has been reported to effectively deliver small interference RNA (siRNA) into cells. Accordingly, a fusion protein of OM and truncated protamine (tp) may be used as a vehicle for the delivery of NgR siRNA into RGC for gene therapy. To test this hypothesis, we constructed OM and tp fusion protein (OM/tp) expression vectors. Using the indirect immunofluorescence labeling method, OM/tp fusion proteins were found to have a high affinity for RGC. The gel shift assay showed that the OM/tp fusion proteins retained the capacity to bind to DNA. Using OM/tp fusion proteins as a delivery tool, the siRNA of NgR was effectively transfected into cells and significantly down-regulated NgR expression levels. More importantly, OM/tp-NgR siRNA dramatically promoted axonal growth of RGC compared with the application of OM/tp recombinant protein or NgR siRNA alone in vitro. In addition, OM/tp-NgR siRNA highly elevated intracellular cyclic adenosine monophosphate (cAMP) levels and inhibited activation of the Ras homolog gene family, member A (RhoA). Taken together, our data demonstrated that the recombinant OM/tp fusion proteins retained the functions of both OM and tp, and that OM/tp-NgR siRNA might potentially be used for the treatment of optic nerve injury.
authors.affiliation: Department of Ophthalmology, Xijing Hospital, Fourth Military Medical University, Xi'an710032, China.
authors.forename: Zhili
authors.initials: Z
authors.lastname: Cui
authors.forename: Jun
authors.initials: J
authors.lastname: Kang
authors.affiliation: Department of Ophthalmology, Xijing Hospital, Fourth Military Medical University, Xi'an710032, China.
authors.forename: Dan
authors.initials: D
authors.lastname: Hu
authors.affiliation: Department of Ophthalmology, Xijing Hospital, Fourth Military Medical University, Xi'an710032, China.
authors.forename: Jian
authors.initials: J
authors.lastname: Zhou
authors.affiliation: Department of Ophthalmology, Xijing Hospital, Fourth Military Medical University, Xi'an710032, China.
authors.forename: Yusheng
authors.initials: Y
authors.lastname: Wang
mesh_terms: D000818:Animals; D001369:Axons / Q000187:drug effects* / Q000378:metabolism; D002135:Calcium-Binding Proteins / Q000235:genetics* / Q000378:metabolism; D000242:Cyclic AMP / Q000378:metabolism; D058851:GPI-Linked Proteins / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism; D005786:Gene Expression Regulation; D018014:Gene Transfer Techniques; D009185:Myelin Proteins / Q000037:antagonists & inhibitors* / Q000235:genetics / Q000378:metabolism; D009416:Nerve Regeneration / Q000187:drug effects; D000070816:Nogo Receptor 1; D010957:Plasmids / Q000737:chemistry; D061251:Primary Cell Culture; D011479:Protamines / Q000235:genetics* / Q000378:metabolism; D034741:RNA, Small Interfering / Q000235:genetics / Q000378:metabolism; D051381:Rats; D011916:Rats, Inbred F344; D011956:Receptors, Cell Surface / Q000037:antagonists & inhibitors* / Q000235:genetics / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D012165:Retinal Ganglion Cells / Q000187:drug effects* / Q000378:metabolism; D015398:Signal Transduction; D020742:rhoA GTP-Binding Protein / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism
chemical_list: D002135:Calcium-Binding Proteins; D058851:GPI-Linked Proteins; D009185:Myelin Proteins; D000070816:Nogo Receptor 1; D011479:Protamines; D034741:RNA, Small Interfering; D011956:Receptors, Cell Surface; D011993:Recombinant Fusion Proteins; C486581:Rtn4r protein, rat; C034733:oncomodulin; D000242:Cyclic AMP; D020742:rhoA GTP-Binding Protein
keywords: Nogo-66 receptor; axon regeneration; oncomodulin; retinal ganglion cells