- Email: [email protected]
Secretion of bacterial chondroitinase ABC from bone marrow stromal cells by glycosylation site mutation: A promising approach for axon regeneration
Medical Hypotheses 77 (2011) 914–916
Contents lists available at SciVerse ScienceDirect
Medical Hypotheses journal homepage: www.elsevier.com/locate/mehy
Secretion of bacterial chondroitinase ABC from bone marrow stromal cells by glycosylation site mutation: A promising approach for axon regeneration Ma Yonggang a,⇑, Liu Min b, Li Yaming a a b
Department of Orthopaedics, Renmin Hospital, Wuhan University, Wuhan City 430060, China Department of Microbiology, Wuhan University School of Medicine, Wuhan City 430071, China
a r t i c l e
i n f o
Article history: Received 7 November 2010 Accepted 6 August 2011
a b s t r a c t Growth-inhibitory chondroitin sulfate proteoglycans (CSPGs) contribute a lot to failure of axon regeneration. Chondroitinase ABC (ChABC) digests glycosaminoglycan chains attached in CSPGs and can thereby promote axonal regeneration beyond a lesion site. However, CSPGs expression are up-regulated for almost 7 weeks after spinal cord injury (SCI) in vivo, so single dose of exogenous ChABC is insufficient for long distance of axon sprout and functional recovery. It is considered an ideal strategy to transfect neurons and/or glia at the injury site with a vector containing the gene encoding chondroitinase, so they can secrete ChABC themselves. Mammalian cells in the current studies, however, can not secret ChABC efficiently. It is well established that glycosylation is a common obstacle for eukaryotic cells to secret bacterial protein. ChABC is a protein heavily glycosylated structurally, and it was reported that inhibiting the glycosylation of xylosyltransferase-1 with a DNA enzyme could reduce GAG chains in the lesion of spinal cord. So presence of glycosylation sites in the bacterial sequence is supposed the barrier that preventing ChABC secretion from mammalian cells. We intend to mutate the key N-glycosylation sites of the bacterial ChABC sequence and transduce it into BMSCs by lentivirus vector. The modified BMSCs are expected to promote axon regeneration through multiple mechanisms, providing sustained ChABC and neurotrophic factors, as well as filling in the cavities formed post-trauma. The transduced BMSCs with gene mutated in key glycosylation sites in the present hypothesis provide a promising strategy to promote axon regeneration. Ó 2011 Elsevier Ltd. All rights reserved.
Introduction Patients with spinal trauma and spinal cord injury (SCI) usually suffer from permanent paralysis for the damaged axons failing to regenerate. The inhibitory microenvironment formed following SCI is considered a major obstacle to axon regeneration, including glial scar and the growth-inhibitory proteins released by degenerating myelin [1–3]. Some cells in the scar zone secret inhibitory molecules [4,5]. Once binding to their specific receptors expressed on the surface of neurons and axons [6], the molecules will prevent axons from sprouting through acting intracellularly via Rho GTPase activation and cytoskeletal modification. A significant contribution to regrowth inhibition in the scar is made by chondroitin sulphate proteoglycans (CSPGs) [7]. These molecules, such as neurocan and NG2, have long sulphated glycosaminoglycan (GAG) chains attached to their core protein component, and the GAG chains are responsible for much of the inhibitory activity [8]. It provides a
⇑ Corresponding author. Address: No. 238, Jiefang Road, Wuchang District, Wuhan City, Hubei Province, China. Tel.: +86 27 88230241; fax: +86 27 88042292. E-mail address: [email protected] (M. Yonggang). 0306-9877/$ - see front matter Ó 2011 Elsevier Ltd. All rights reserved. doi:10.1016/j.mehy.2011.08.010
promising approach to promote axons to regenerate by targeting the inhibitory GAG chains.
Bacterial ChABC can degrade CSPGs and promote axon sprouting but its efficacy is limited by administration methods Chondroitinase ABC is a bacterial enzyme that degrades these inhibitory carbohydrate chains. It promotes axon regeneration in rats, as demonstrated first in the nigrostriatal tract [9] and also in the spinal cord [10]. It is active when injected into the mammalian CNS, as it depletes GAG immunoreactivity surrounding an injury site and concomitantly generates carbohydrate products which are not seen in normal tissue [10]. Chondroitinase may promote recovery by other mechanisms, including dispersal of other axon-inhibitory molecules, neuroprotection [11], sprouting and synaptic plasticity in spared pathways due to removal of perineuronal nets [12]. Whatever the predominant mechanism, chondroitinase is clearly a promising treatment for spinal cord injury. CSPG-immunoreactivity (IR) in the injury site remained high for at least 49 days after spinal cord contusion [13]. ChABC, however, keeps its enzymatic activity for only a few hours at 37 °C [14]. So it is not effective enough for single dose of exogenous ChABC to
M. Yonggang et al. / Medical Hypotheses 77 (2011) 914–916
block the CSPGs secreted sustainedly following SCI. Tom et al. reported that administration of chondroitinase ABC rostral or caudal to a spinal cord injury site promoted significant sprouting of 5HT + fibers but not functional recovery [15]. Repeated injections or intrathecal catheter could provide bacterial ChABC for a long period of time and thus promote extensive regeneration of both corticospinal and sensory axons, accompanied by significant functional motor recovery [10,16]. But chronic delivery is invasive, infection-prone, clinically problematic and even induce immunogenicity. It would be an ideal strategy to transfect neurons and/ or glia at the injury site with a vector containing the gene for chondroitinase, so they can secrete the enzyme themselves. Presence of glycosylation sites in bacterial sequence is supposed the barrier that limits ChABC secretion from mammalian cells Gene encoding bacterial ChABC was cloned and transduced into bone marrow stromal cells (BMSCs) with lentivirus vector in our preliminary study. Though a eukaryotic signal sequence from mouse matrix metalloprotease-9 was added to the 50 end to allow recognition by eukaryotic ribosomes, the expression of ChABC was detected intracellularly by Western blot assay but not in the supernatant of cultured BMSCs. This work extends our knowledge about the conditions required to express bacterial proteins in eukaryotic cells. The same question also puzzled other researchers in this field. Cafferty et al. [17] provided a transgenic test of the role of ChABC in transgenic mice, using GFAP promoter and the original signal sequence of Proteus to express it in astrocytes. As a result, efficient secretion of the enzyme was not proven and the immunoreaction tostub antibody was limited several millimeters from the center of the lesion. Moreover, the astrocytic expression was not sufficient to allow corticospinal axons to regenerate beyond the lesion. Curinga et al. [18] have recently reported expression of a different bacterial enzyme, chondroitinase AC, in mammalian cells, using an immunoglobulin signal sequence and adenovirus vector. However the specific activity of the secreted product was low. There must be some cryptic signals, such as glycosylation, that may be recognised in bacterial sequences and play a role as obstacle to ChABC secretion from transfected mammalian cells. The research of Grimpe et al. with a newly designed DNA enzyme appeared to confirm this hypothesis [19]. The DNA enzyme, which targets the mRNA of a critical enzyme that initiates glycosylation of xylosyltransferase-1, could reduce GAG chains in TGF-betastimulated astrocytes in culture and in the lesion of spinal cord. Recently Muir and his colleagues confirmed this hypothesis [20]. Chondroitinase ABC was heavily glycosylated when expressed in mammalian cells or in a mammalian translation system, and this process prevented secretion of functional enzyme. Directed mutagenesis of selected N-glycosylation sites allowed efficient secretion of active chondroitinase. BMSCs transfecting with gene mutated in key N-glycosylation sites are supposed to secret ChABC efficiently and promote axonal regrowth through multiple ways. We intend to mutate the key N-glycosylation sites of the bacterial ChABC sequence so that mammalian cells can produce and secrete it. The mutated sequence is then transduced into BMSCs by lentivirus vector. The modified BMSCs are expected to secret active ChABC for a longer period in culture and promote axons to sprout when transplanted into a lesion site in spinal cord. There is great technical difficulty in transfecting neurons clinically, which would aggravate the injury. BMSCs are chosen as the transfection vector also for their other properties. BMSCs have been confirmed that they can differentiate into neuron-like cells under experimental cell culture conditions [21]. Even they formed ‘‘guiding strands’’ for host axonal growth and promoted partial functional recovery after grafting to sites of spinal cord injury. Though it remains
915
controversial whether these cells can form neural cells when exposed to the CNS microenvironment [22], BMSCs could at least fill in the cavities formed post-trauma and thus inhibit the glial scar. BMSCs secret two neurotrophins when cultured in vitro, NGF and BDNF [23], which are required for repair of neural tissues. BMSCs can still secret BDNF during culture in vitro though undergoing some gene manipulation [24]. If the hypothesis is confirmed, the BMSCs with sequence mutated in glycosalation sites could secret BDNF, and more interestingly, active ChABC for a long period, and thus promote axon regeneration and neurological recovery efficiently. Even the microenvironment is unfavorable for them to differentiate into neurons, they could fill the cavities formed following tissue necrosis when they are transplanted into the injury site. Conflict of interest statement None declared. Acknowledgments The study is supported by Health Department of Hubei Province, China (Grant No. 20101018). References [1] Prinjha R, Moore SE, Vinson M, Blake S, Morrow R, Christie G. Inhibitor of neurite outgrowth in humans. Nature 2000;403:383–4. [2] Chen MS, Huber AB, van der Haar ME, et al. Nogo-A is a myelin-associated neurite outgrowth inhibitor and an antigen for monoclonal antibody IN-1. Nature 2000;403:434–9. [3] Kottis V, Thibault P, Mikol D, Xiao ZC, Zhang R, Dergham P. Oligodendrocytemyelin glycoprotein (OMgp) is an inhibitor of neurite outgrowth. J Neurochem 2002;82:1566–9. [4] Silver JS, Miller JH. Regeneration beyond the glial scar. Nat Rev Neurosci 2004;5:146–61. [5] Yiu G, He Z. Glial inhibition of CNS axon regeneration. Nat Rev Neurosci 2006;7:617–27. [6] Shen Y, Tenney AP, Busch SA, et al. PTPsigma is a receptor for chondroitin sulfate proteoglycan, an inhibitor of neural regeneration. Science 2009;326: 592–6. [7] Kwok JC, Afshari F, García-Alías G, Fawcett JW. Proteoglycans in the central nervous system: plasticity, regeneration and their stimulation with chondroitinase ABC. Restor Neurol Neurosci 2008;26:131–45. [8] Huang DW, McKerracher L, Braun PE, David S. A therapeutic vaccine approach to stimulate axon regeneration in the adult mammalian spinal cord. Neuron 1999;24(3):639–47. [9] Moon LD, Asher RA, Rhodes KE, Fawcett JW. Regeneration of CNS axons back to their target following treatment of adult brain with chondroitinase ABC. Nat Neurosci 2001;4:465–6. [10] Bradbury EJ, Moon LD, Popat RJ, et al. Chondroitinase ABC promotes functional recovery after spinal cord injury. Nature 2002;416:589–90. [11] Carter LM, Starkey ML, Akrimi SF, Davies M, McMahon SB, Bradbury EJ. The yellow fluorescent protein (YFP-H) mouse reveals neuroprotection as a novel mechanism underlying chondroitinase ABC-mediated repair after spinal cord injury. J Neurosci 2008;28:14107–20. [12] Crespo D, Asher RA, Lin R, Rhodes KE, Fawcett JW. How does chondroitinase promote functional recovery in the damaged CNS? Exp Neurol 2007;206: 159–71. [13] Iseda T, Okuda T, Kane-Goldsmith N, et al. Single, high-dose intraspinal injection of chondroitinase reduces glycosaminoglycans in injured spinal cord and promotes corticospinal axonal regrowth after hemisection but not contusion. J Neurotrauma 2008;25:334–49. [14] Lee H, McKeon RJ, Bellamkonda RV. Sustained delivery of thermostabilized chABC enhances axonal sprouting and functional recovery after spinal cord injury. Proc Natl Acad Sci USA 2010;107:3340–5. [15] Tom VJ, Kadakia R, Santi L, Houlé JD. Administration of chondroitinase ABC rostral or caudal to a spinal cord injury site promotes anatomical but not functional plasticity. J Neurotrauma 2009;26:2323–33. [16] Huang WC, Kuo WC, Hsu SH, Cheng CH, Liu JC, Cheng H. Gait analysis of spinal cord injured rats after delivery of chondroitinase ABC and adult olfactory mucosa progenitor cell transplantation. Neurosci Lett 2010;472:79–84. [17] Cafferty WB, Yang SH, Duffy PJ, Li S, Strittmatter SM. Functional axonal regeneration through astrocytic scar genetically modified to digest CSPGs. J Neurosci 2007;27:2176–85. [18] Curinga GM, Snow DM, Mashburn C, et al. Mammalian-produced chondroitinase AC mitigates axon inhibition by chondroitin sulfate proteoglycans. J Neurochem 2007;102:275–88.
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M. Yonggang et al. / Medical Hypotheses 77 (2011) 914–916
[19] Grimpe B, Silver J. A novel DNA enzyme reduces glycosaminoglycan chains in the glial scar and allows microtransplanted dorsal root ganglia axons to regenerate beyond lesions in the spinal cord. J Neurosci 2004;24:1393–7. [20] Muir EM, Fyfe I, Gardiner S, et al. Modification of N-glycosylation sites allows secretion of bacterial chondroitinase ABC from mammalian cells. J Biotechnol 2010;145(2):103–10. [21] Woodbury D, Schwarz EJ, Prockop DJ, Black IB. Adult rat and human bone marrow stromal cells differentiate into neurons. J Neurosci Res 2000;61: 364–70.
[22] Rismanchi N, Floyd CL, Berman RF, Lyeth BG. Cell death and long-term maintenance of neuron-like state after differentiation of rat bone marrow stromal cells: a comparison of protocols. Brain Res 2003;991:46–55. [23] Yaghoobi MM, Mowla SJ. Differential gene expression pattern of neurotrophins and their receptors during neuronal differentiation of rat bone marrow stromal cells. Neurosci Lett 2006;397:149–54. [24] Liu M, Ma Y. Expression of soluble Nogo-66 receptor and brain-derived neurotrophic factor in transduced rat bone marrow stromal cells. J Clin Neurosci 2010;17:762–5.
Contents lists available at SciVerse ScienceDirect
Medical Hypotheses journal homepage: www.elsevier.com/locate/mehy
Secretion of bacterial chondroitinase ABC from bone marrow stromal cells by glycosylation site mutation: A promising approach for axon regeneration Ma Yonggang a,⇑, Liu Min b, Li Yaming a a b
Department of Orthopaedics, Renmin Hospital, Wuhan University, Wuhan City 430060, China Department of Microbiology, Wuhan University School of Medicine, Wuhan City 430071, China
a r t i c l e
i n f o
Article history: Received 7 November 2010 Accepted 6 August 2011
a b s t r a c t Growth-inhibitory chondroitin sulfate proteoglycans (CSPGs) contribute a lot to failure of axon regeneration. Chondroitinase ABC (ChABC) digests glycosaminoglycan chains attached in CSPGs and can thereby promote axonal regeneration beyond a lesion site. However, CSPGs expression are up-regulated for almost 7 weeks after spinal cord injury (SCI) in vivo, so single dose of exogenous ChABC is insufficient for long distance of axon sprout and functional recovery. It is considered an ideal strategy to transfect neurons and/or glia at the injury site with a vector containing the gene encoding chondroitinase, so they can secrete ChABC themselves. Mammalian cells in the current studies, however, can not secret ChABC efficiently. It is well established that glycosylation is a common obstacle for eukaryotic cells to secret bacterial protein. ChABC is a protein heavily glycosylated structurally, and it was reported that inhibiting the glycosylation of xylosyltransferase-1 with a DNA enzyme could reduce GAG chains in the lesion of spinal cord. So presence of glycosylation sites in the bacterial sequence is supposed the barrier that preventing ChABC secretion from mammalian cells. We intend to mutate the key N-glycosylation sites of the bacterial ChABC sequence and transduce it into BMSCs by lentivirus vector. The modified BMSCs are expected to promote axon regeneration through multiple mechanisms, providing sustained ChABC and neurotrophic factors, as well as filling in the cavities formed post-trauma. The transduced BMSCs with gene mutated in key glycosylation sites in the present hypothesis provide a promising strategy to promote axon regeneration. Ó 2011 Elsevier Ltd. All rights reserved.
Introduction Patients with spinal trauma and spinal cord injury (SCI) usually suffer from permanent paralysis for the damaged axons failing to regenerate. The inhibitory microenvironment formed following SCI is considered a major obstacle to axon regeneration, including glial scar and the growth-inhibitory proteins released by degenerating myelin [1–3]. Some cells in the scar zone secret inhibitory molecules [4,5]. Once binding to their specific receptors expressed on the surface of neurons and axons [6], the molecules will prevent axons from sprouting through acting intracellularly via Rho GTPase activation and cytoskeletal modification. A significant contribution to regrowth inhibition in the scar is made by chondroitin sulphate proteoglycans (CSPGs) [7]. These molecules, such as neurocan and NG2, have long sulphated glycosaminoglycan (GAG) chains attached to their core protein component, and the GAG chains are responsible for much of the inhibitory activity [8]. It provides a
⇑ Corresponding author. Address: No. 238, Jiefang Road, Wuchang District, Wuhan City, Hubei Province, China. Tel.: +86 27 88230241; fax: +86 27 88042292. E-mail address: [email protected] (M. Yonggang). 0306-9877/$ - see front matter Ó 2011 Elsevier Ltd. All rights reserved. doi:10.1016/j.mehy.2011.08.010
promising approach to promote axons to regenerate by targeting the inhibitory GAG chains.
Bacterial ChABC can degrade CSPGs and promote axon sprouting but its efficacy is limited by administration methods Chondroitinase ABC is a bacterial enzyme that degrades these inhibitory carbohydrate chains. It promotes axon regeneration in rats, as demonstrated first in the nigrostriatal tract [9] and also in the spinal cord [10]. It is active when injected into the mammalian CNS, as it depletes GAG immunoreactivity surrounding an injury site and concomitantly generates carbohydrate products which are not seen in normal tissue [10]. Chondroitinase may promote recovery by other mechanisms, including dispersal of other axon-inhibitory molecules, neuroprotection [11], sprouting and synaptic plasticity in spared pathways due to removal of perineuronal nets [12]. Whatever the predominant mechanism, chondroitinase is clearly a promising treatment for spinal cord injury. CSPG-immunoreactivity (IR) in the injury site remained high for at least 49 days after spinal cord contusion [13]. ChABC, however, keeps its enzymatic activity for only a few hours at 37 °C [14]. So it is not effective enough for single dose of exogenous ChABC to
M. Yonggang et al. / Medical Hypotheses 77 (2011) 914–916
block the CSPGs secreted sustainedly following SCI. Tom et al. reported that administration of chondroitinase ABC rostral or caudal to a spinal cord injury site promoted significant sprouting of 5HT + fibers but not functional recovery [15]. Repeated injections or intrathecal catheter could provide bacterial ChABC for a long period of time and thus promote extensive regeneration of both corticospinal and sensory axons, accompanied by significant functional motor recovery [10,16]. But chronic delivery is invasive, infection-prone, clinically problematic and even induce immunogenicity. It would be an ideal strategy to transfect neurons and/ or glia at the injury site with a vector containing the gene for chondroitinase, so they can secrete the enzyme themselves. Presence of glycosylation sites in bacterial sequence is supposed the barrier that limits ChABC secretion from mammalian cells Gene encoding bacterial ChABC was cloned and transduced into bone marrow stromal cells (BMSCs) with lentivirus vector in our preliminary study. Though a eukaryotic signal sequence from mouse matrix metalloprotease-9 was added to the 50 end to allow recognition by eukaryotic ribosomes, the expression of ChABC was detected intracellularly by Western blot assay but not in the supernatant of cultured BMSCs. This work extends our knowledge about the conditions required to express bacterial proteins in eukaryotic cells. The same question also puzzled other researchers in this field. Cafferty et al. [17] provided a transgenic test of the role of ChABC in transgenic mice, using GFAP promoter and the original signal sequence of Proteus to express it in astrocytes. As a result, efficient secretion of the enzyme was not proven and the immunoreaction tostub antibody was limited several millimeters from the center of the lesion. Moreover, the astrocytic expression was not sufficient to allow corticospinal axons to regenerate beyond the lesion. Curinga et al. [18] have recently reported expression of a different bacterial enzyme, chondroitinase AC, in mammalian cells, using an immunoglobulin signal sequence and adenovirus vector. However the specific activity of the secreted product was low. There must be some cryptic signals, such as glycosylation, that may be recognised in bacterial sequences and play a role as obstacle to ChABC secretion from transfected mammalian cells. The research of Grimpe et al. with a newly designed DNA enzyme appeared to confirm this hypothesis [19]. The DNA enzyme, which targets the mRNA of a critical enzyme that initiates glycosylation of xylosyltransferase-1, could reduce GAG chains in TGF-betastimulated astrocytes in culture and in the lesion of spinal cord. Recently Muir and his colleagues confirmed this hypothesis [20]. Chondroitinase ABC was heavily glycosylated when expressed in mammalian cells or in a mammalian translation system, and this process prevented secretion of functional enzyme. Directed mutagenesis of selected N-glycosylation sites allowed efficient secretion of active chondroitinase. BMSCs transfecting with gene mutated in key N-glycosylation sites are supposed to secret ChABC efficiently and promote axonal regrowth through multiple ways. We intend to mutate the key N-glycosylation sites of the bacterial ChABC sequence so that mammalian cells can produce and secrete it. The mutated sequence is then transduced into BMSCs by lentivirus vector. The modified BMSCs are expected to secret active ChABC for a longer period in culture and promote axons to sprout when transplanted into a lesion site in spinal cord. There is great technical difficulty in transfecting neurons clinically, which would aggravate the injury. BMSCs are chosen as the transfection vector also for their other properties. BMSCs have been confirmed that they can differentiate into neuron-like cells under experimental cell culture conditions [21]. Even they formed ‘‘guiding strands’’ for host axonal growth and promoted partial functional recovery after grafting to sites of spinal cord injury. Though it remains
915
controversial whether these cells can form neural cells when exposed to the CNS microenvironment [22], BMSCs could at least fill in the cavities formed post-trauma and thus inhibit the glial scar. BMSCs secret two neurotrophins when cultured in vitro, NGF and BDNF [23], which are required for repair of neural tissues. BMSCs can still secret BDNF during culture in vitro though undergoing some gene manipulation [24]. If the hypothesis is confirmed, the BMSCs with sequence mutated in glycosalation sites could secret BDNF, and more interestingly, active ChABC for a long period, and thus promote axon regeneration and neurological recovery efficiently. Even the microenvironment is unfavorable for them to differentiate into neurons, they could fill the cavities formed following tissue necrosis when they are transplanted into the injury site. Conflict of interest statement None declared. Acknowledgments The study is supported by Health Department of Hubei Province, China (Grant No. 20101018). References [1] Prinjha R, Moore SE, Vinson M, Blake S, Morrow R, Christie G. Inhibitor of neurite outgrowth in humans. Nature 2000;403:383–4. [2] Chen MS, Huber AB, van der Haar ME, et al. Nogo-A is a myelin-associated neurite outgrowth inhibitor and an antigen for monoclonal antibody IN-1. Nature 2000;403:434–9. [3] Kottis V, Thibault P, Mikol D, Xiao ZC, Zhang R, Dergham P. Oligodendrocytemyelin glycoprotein (OMgp) is an inhibitor of neurite outgrowth. J Neurochem 2002;82:1566–9. [4] Silver JS, Miller JH. Regeneration beyond the glial scar. Nat Rev Neurosci 2004;5:146–61. [5] Yiu G, He Z. Glial inhibition of CNS axon regeneration. Nat Rev Neurosci 2006;7:617–27. [6] Shen Y, Tenney AP, Busch SA, et al. PTPsigma is a receptor for chondroitin sulfate proteoglycan, an inhibitor of neural regeneration. Science 2009;326: 592–6. [7] Kwok JC, Afshari F, García-Alías G, Fawcett JW. Proteoglycans in the central nervous system: plasticity, regeneration and their stimulation with chondroitinase ABC. Restor Neurol Neurosci 2008;26:131–45. [8] Huang DW, McKerracher L, Braun PE, David S. A therapeutic vaccine approach to stimulate axon regeneration in the adult mammalian spinal cord. Neuron 1999;24(3):639–47. [9] Moon LD, Asher RA, Rhodes KE, Fawcett JW. Regeneration of CNS axons back to their target following treatment of adult brain with chondroitinase ABC. Nat Neurosci 2001;4:465–6. [10] Bradbury EJ, Moon LD, Popat RJ, et al. Chondroitinase ABC promotes functional recovery after spinal cord injury. Nature 2002;416:589–90. [11] Carter LM, Starkey ML, Akrimi SF, Davies M, McMahon SB, Bradbury EJ. The yellow fluorescent protein (YFP-H) mouse reveals neuroprotection as a novel mechanism underlying chondroitinase ABC-mediated repair after spinal cord injury. J Neurosci 2008;28:14107–20. [12] Crespo D, Asher RA, Lin R, Rhodes KE, Fawcett JW. How does chondroitinase promote functional recovery in the damaged CNS? Exp Neurol 2007;206: 159–71. [13] Iseda T, Okuda T, Kane-Goldsmith N, et al. Single, high-dose intraspinal injection of chondroitinase reduces glycosaminoglycans in injured spinal cord and promotes corticospinal axonal regrowth after hemisection but not contusion. J Neurotrauma 2008;25:334–49. [14] Lee H, McKeon RJ, Bellamkonda RV. Sustained delivery of thermostabilized chABC enhances axonal sprouting and functional recovery after spinal cord injury. Proc Natl Acad Sci USA 2010;107:3340–5. [15] Tom VJ, Kadakia R, Santi L, Houlé JD. Administration of chondroitinase ABC rostral or caudal to a spinal cord injury site promotes anatomical but not functional plasticity. J Neurotrauma 2009;26:2323–33. [16] Huang WC, Kuo WC, Hsu SH, Cheng CH, Liu JC, Cheng H. Gait analysis of spinal cord injured rats after delivery of chondroitinase ABC and adult olfactory mucosa progenitor cell transplantation. Neurosci Lett 2010;472:79–84. [17] Cafferty WB, Yang SH, Duffy PJ, Li S, Strittmatter SM. Functional axonal regeneration through astrocytic scar genetically modified to digest CSPGs. J Neurosci 2007;27:2176–85. [18] Curinga GM, Snow DM, Mashburn C, et al. Mammalian-produced chondroitinase AC mitigates axon inhibition by chondroitin sulfate proteoglycans. J Neurochem 2007;102:275–88.
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M. Yonggang et al. / Medical Hypotheses 77 (2011) 914–916
[19] Grimpe B, Silver J. A novel DNA enzyme reduces glycosaminoglycan chains in the glial scar and allows microtransplanted dorsal root ganglia axons to regenerate beyond lesions in the spinal cord. J Neurosci 2004;24:1393–7. [20] Muir EM, Fyfe I, Gardiner S, et al. Modification of N-glycosylation sites allows secretion of bacterial chondroitinase ABC from mammalian cells. J Biotechnol 2010;145(2):103–10. [21] Woodbury D, Schwarz EJ, Prockop DJ, Black IB. Adult rat and human bone marrow stromal cells differentiate into neurons. J Neurosci Res 2000;61: 364–70.
[22] Rismanchi N, Floyd CL, Berman RF, Lyeth BG. Cell death and long-term maintenance of neuron-like state after differentiation of rat bone marrow stromal cells: a comparison of protocols. Brain Res 2003;991:46–55. [23] Yaghoobi MM, Mowla SJ. Differential gene expression pattern of neurotrophins and their receptors during neuronal differentiation of rat bone marrow stromal cells. Neurosci Lett 2006;397:149–54. [24] Liu M, Ma Y. Expression of soluble Nogo-66 receptor and brain-derived neurotrophic factor in transduced rat bone marrow stromal cells. J Clin Neurosci 2010;17:762–5.