Acta Neurol Belg DOI 10.1007/s13760-015-0425-0
REVIEW ARTICLE
Axonal regeneration inhibitors: emerging therapeutic options T. W. Rosochowicz • S. Wrotek • W. Kozak
Received: 22 September 2014 / Accepted: 30 December 2014 Ó Belgian Neurological Society 2015
Abstract For the most part, the central nervous system is unable to regenerate. The majority of injuries of vascular, inflammatory, degenerative and traumatic aetiology lead to an irreversible loss of central nervous system function. The paper presents the role of Nogo-A, MAG and OMgp proteins in the inhibition of central nervous system regeneration, and their potential clinical significance. Keywords Nogo-A MAG OMgp Regeneration of the central nervous system
takes some time. Some proteins, including Nogo, myelinassociated glyocoprotein (MAG) and oligodendrocyte myelin glycoprotein (OMgp), are released at the moment of injury of the oligodendrocytes and myelin sheaths. Those proteins demonstrate strong axonal regeneration inhibiting properties. It is believed that they play a major role in the inhibition of regeneration of the CNS. The purpose of this paper is to provide a brief presentation of the myelinassociated proteins that inhibit regeneration of axons, as well as some of their inhibitors, applied in preliminary animal models and in first human clinical trials.
Introduction Axon regeneration scheme In addition to some structures of the hypothalamus and neurons localised within the olfactory bulb, the central nervous system (CNS) is depleted of regenerative properties. Therefore, obvious and significant regeneration of cerebral and spinal neurons that could restore the function lost in the course of various neurological conditions, including cerebral strokes, cerebro-cranial injuries, neuroinfections, sclerosis multiplex and broadly understood neurodegenerative diseases, has not been observed. In contrast to the CNS, the peripheral nervous system presents some significant regenerative properties. Injury of the CNS is associated with damage to neurons and glial cells. Depending on the aetiological factor, the injury may be of mechanical, ischaemic, immunological or inflammatory origin. A glial scar is gradually formed, constituting a physical barrier and containing substances that inhibit regeneration of neurons. However, glial scar formation T. W. Rosochowicz (&) S. Wrotek W. Kozak Nicolaus Copernicus University, Torun´, Poland e-mail: [email protected]
It was experimentally demonstrated that, following an injury, regenerative growth cones appear on the ends of axons [1]. Ultrastructural evaluation revealed the presence of lamellar structures, referred to as lamellipodia, possessing smaller outgrowths, known as filopodia. When axon continuity is compromised in favourable conditions, i.e., in presence of Schwann cells, it creates a necessary scaffolding allowing regeneration of the axon. That phenomenon was already demonstrated in experimental studies at the beginning of the 20th century [2]. If stimulating factors are present, the axon begins to grow. In the peripheral nervous system, complete or partial axon function restoration occurs following an injury, depending on its type (e.g., according to Seddon-Sunderland classifications of 1943 and 1957). The central nervous system has no regenerative properties, because of the presence of inhibitors of the axonal regeneration. At the moment of injury, those inhibitors are released from myelin. Similar properties are also demonstrated by a newly forming glial scar. In early 1989, an experiment was conducted [3] demonstrating that non-
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myelinised nerve fibres present in the CNS have the ability to regenerate following a chemical axonotomy. However, the process does not occur in the neighbourhood of the myelinised fibres. The presence of myelin leads to the release of regeneration inhibiting factors, resulting in decay of the fibres.
(GTP-ase) RhoA activates a Rho-dependent kinase (ROCK) [10]. That activation leads to increased rigidity of the actin cytoskeleton resulting in inhibition of the axonal growth cone and in its further elongation. Calcium ions are another secondary messenger. An increase in their intracellular level leads to reduced cAMP concentration, and that phenomenon causes inhibition of axonal regeneration (see Fig. 1).
The glial scar Within approximately 14 days following an injury to the central nervous system, a glial scar is formed preventing axons from growing [4]. The scar is formed of fibroblasts originating from meningeal cells, astrocytes, oligodendrocytes and microglia. Those cells aggregate at the site of an injury. The process has two implications: it creates a physical barrier hindering penetration of budding axons or even preventing the penetration completely, and the forming layer constitutes a source of substances, such as of proteoglycans chondroitin sulphate and keratan sulphate, which inhibit axonal regeneration [5]. Those compounds possess some very strong inhibiting properties that impact nervous system regeneration. In addition, a major role is played by the myelin-associated axonal regeneration inhibitors described below [6]. Proteoglycans are huge molecules abundant in the human organism. They are composed of a protein core covalently bound to chains of glucosaminoglycans, e.g., heparan sulphate. The enzymechondroitinase [7] separates those chains, thereby depleting the axon regeneration inhibiting properties of those compounds.
Myelin-associated inhibitors of axonal regeneration Myelin contains three important proteins responsible for inhibition of axonal regeneration: Nogo-A, MAG and OMgp, previously known as arretin. All three of these proteins have a common NgR1 receptor (NgR-Nogo receptor). MAG also binds to a second subtype of that receptor: NgR2. The NgR1 receptor is a GPI-anchored protein. Glycosylphosphatidylinositol (GPI) is a glycolipid allowing protein anchoring in so-called lipid rafts that binds to a protein C-terminal. Formation of the complex is necessary for activation of the receptor. The literature reports other subunits of that complex: p75NTR (low affinity neurotrophin receptor) [8], TROY (sometimes referred to as TAJ) and LINGO-1 (leucine-rich repeat and immunoglobulin-like domain-containing Nogo receptorinteracting protein) [9]. A signal may be transmitted further only after the complex is formed. There are two secondary messengers: RhoA and an increase in the concentration of intracellular calcium ions. Guanozino-50 triphosphatase
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Nogo An anti-myelin monoclonal antibody IN-1 was successfully created in 1988 [11]. The antibody was produced in order to block the axonal regeneration inhibiting properties of the myelin. Later it was demonstrated that the IN-1 monoclonal antibody is targeted at Nogo-A [11]. IgM class antibodies were used in the 1970s, and more recent studies employ IgG class antibodies. Several years later, in 2000 [12], the Nogo gene was successfully cloned, and its principal product, Nogo-A, was demonstrated to be the proper antigen for the IN-1 antibody. Nogo is one of the most important and the best-studied proteins responsible for the inhibition of axon growth in an injured central nervous system. In the nervous system, Nogo-A is a membranous protein present principally in oligodendrocytes and myelin sheaths. Moreover, the Nogo family includes several isoforms, Nogo-B and Nogo-C, which play no significant role in regeneration of the nervous system. Nogo-B is present in various tissues and Nogo-C is principally encountered in muscular tissue [13]. Each of those three proteins is coded by the same Nogo gene. They all share the same C-terminal chain of 188 amino-acids. Due to that amino-acid sequence Nogo proteins may be included in a small family of reticulon proteins (Nogo-A is also referred to as RTN4), present in the nervous system. Nogo proteins are characterised by the presence of two hydrophobic sections connected with a loop consisting of 66 amino-acids. Studies have indicated that those hydrophobic sections demonstrated particular axon regeneration inhibiting properties. Therefore, the sequence is referred to as ‘‘Nogo-66’’ [14]. It is present in all isoforms of the Nogo protein. It demonstrates a high affinity to the NgR receptor. The Nogo-A isoform, and its specific N-terminus, is recognised by the monoclonal antibody IN-1 [15]. It is notable that the Nogo-A protein is absent in some animals, including salamanders [16] and fish that possess some astounding regenerative properties, indicating that it is involved in regeneration of some body parts (such as tails) and, therefore, also of the spinal cord. Thus, there is important evolutionary evidence for the inhibitory effect of the Nogo-A protein on axonal regeneration in the central nervous system (see Fig. 2).
Acta Neurol Belg Fig. 1 Action mechanisms of myelin-associated inhibitors of axonal regeneration on receptors (according to Schwab 2005)
Fig. 2 Nogo protein isoforms
Myelin-associated glycoprotein (MAG) Myelin-associated glycoprotein (MAG) is one the first proteins discovered to be able to inhibit axonal regeneration [17]. MAG is present in the periaxonal space and plays
a significant role in mutual myelin-axon interaction. For example, it is associated with the onset of myelinisation, maintenance of myelin and myelin sheath integrity. MAG has double properties; it is an axonal growth factor but it also inhibits axonal regeneration. MAG has an effect on
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neuron changes during ontogenesis; during the foetal period, it is a stimulant and when myelinisation is complete, MAG changes into a suppressant [18].
receptor type B (LILRB2). The NgR receptor is associated with complex-forming subunits: p75, LINGO-1 and TROY [21]. As previously mentioned, the Rho-A/ROCK system is an effector for the NgR complex (see Fig. 3).
Oligodendrocyte myelin glycoprotein (OMgp) Clinical significance As indicated by its name, OMgp is localised principally in oligodendrocytes, but it is also encountered in neurons. Via glycophosphatydylinositol it is anchored in the plasmic membrane. This protein inhibits axonal regeneration [19].
Receptors of axonal regeneration inhibitors The NgR receptor (proper name NgR1) is a glycoprotein anchored in the plasmic membrane with glycophosphatydylinositol. The receptor demonstrates affinity towards Nogo-A, MAG and OMgp. The NgR2 isoform of the receptor also binds to MAG. Another recently discovered receptor, paired immunoglobulin-like receptor type B (PirB) [20], also shows affinity to all the myelin-associated proteins mentioned above, thereby inhibiting axonal regeneration. Even blocking the NgR receptor could not restore the regenerative properties of axons located in the central nervous system. That phenomenon is most probably associated with the presence of the other receptor, PirB. That receptor was discovered in animal studies. In humans, a similar role is played by leukocytic immunoglobulin-like
Fig. 3 The NgR1 receptor activation mechanism (according to Oertle 2003)
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Nogo extracellular peptide, residues 1-40 (NEP 1-40) is a competitive agonist of the NgR receptor [22]. In experimental injury of the spinal cord, intrathecal administration of NEP1-40 [23] clearly improved regeneration of axons in the spinal cord. Of course, use of the peptide in acute spinal cord injury may be difficult. Considering that fact, Li [24] studied subcutaneous administration of the NEP 1-40 peptide. Even administration of the study substance a week after an injury proved not to be inferior compared to administration of the substance at the moment of the spinal cord injury. The study demonstrated an intensified axonal growth in the cortical-medullary pathways and ‘‘budding’’ of serotoninergic fibres, as well as reconstruction of synapses. Improvement of motor function of paralysed muscular groups was also achieved. The Rho protein family [25] is characterised by the presence of guanosine-50 triphosphatase (GTP-ase)-containing domains. Due to their presence, the proteins are referred to as molecular switches. Each of those proteins may be in the active GTP (guanosine-50 triphosphate)-containing form or the inactive GDP (guanosine-50 diphosphate)-containing
Acta Neurol Belg Fig. 4 Mechanism of action of selected compounds restoring axonal regeneration
form. The p75NTR subunit releases Rho from the Rho-GDI (guanine nucleotide dissociation inhibitor) complex and inhibits transition from the inactive to the active form [26]. ROCK is another protein activated by Rho-A. Considering that fact, it was assumed that inhibition of the Rho-A/ROCK system may lead to secondary improvement of axonal regeneration in the CNS. The Rho kinase inhibitor known as Y-27632 may be an example of a potential pharmacological block of the Rho-A/ROCK system. Its mechanism of action consists in competitive binding to the kinase at the ATPbinding site. A similar role is played by a bacterial (obtained from Clostridium botulinum) endotoxin transferase C3, which is also able to inhibit Rho via the process of ADP ribosylation. In a comparative study, Dergham [27] demonstrated the superior efficiency of C3 transferase compared to Y-27632. Both proteins also demonstrate neuroprotective properties. Fournier’s experiments [28] on an animal model demonstrated that application of Y-27632 enabled and improved regeneration of cortical-medullary pathways, and speeded up the return of the motor activity. The C3 transferase proved to be less efficient in that study. A short Pep5 peptide is another compound that is potentially able to support regeneration of the nervous system is. The peptide binds at the site of connection between p75NTR and Rho-GDI. Its efficacy was demonstrated as it allowed regeneration of neurons in spinal ganglions and in the cerebellum [29]. In the US, clinical trials are currently being carried out with the C3 transferase
analogue BA-210 (Cethrin), administered intrathecally to patients with acute cervical and thoracic spine injury. Rolipram—a phosphodiesterase 4 inhibitor—is another promising medicinal product. The drug is used principally to treat depressive syndrome, and it demonstrates antipsychotic properties as well as anti-inflammatory properties. It was also demonstrated that, by increasing the intracellular cAMP level, the drug improved the regenerative properties of neurons [30]. Dantrolene is a compound that inhibits intracellular calcium ion release. Administration of the drug blocks Ca2?, thus suppressing NI-35 (neuron growth inhibitor). That prevents degeneration of a growth cone [31]. Experimental studies demonstrated that Riluzol, approved for treating SLA, may also be applicable to spinal cord injuries, improving re-innervation even if administered several days after the injury [32] (see Fig. 4).
Conclusion A brief description of the proteins that inhibit axonal regeneration, the role of glial scars and of the Rho-A/ ROCK system presented in this paper indicate that potential possibilities exist for reversing of the currently ‘‘irreversible’’ damage of the CNS. Although only selected studies have been presented, the majority of them were completed on animal models. However, as the first clinical
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trials with humans emerge, using classical methods of neurological therapy and modern neurosurgical procedures, the process of ‘‘irreversible’’ injury of the CNS will soon be successfully reversed. Acknowledgments
This study received no financial support.
Conflict of interest The authors report no financial or other conflict of interest associated with this study.
References 1. Schwab JM, Failli V, Chetodal A (2005) Injury-related dynamic myelin/oligodendrocyte axon-outgrowth inhibition in the central nervous system. Lancet 365:2055–2057 2. Tello Y (1911) La influencia del neurotropismo en la regeneracion de los centros nerviosos. Trab Lab Invest Biol 9:123–159 3. Berry M (1982) Post-injury myelin-breakdown products inhibit axonal growth: an hypothesis to explain the failure of axonal regeneration in the mammalian central nervous system. Bibliotheca Anatomica 23:1–11 4. Silver J, Miller JH (2004) Regeneration beyond the glial scar. Nat Rev Neurosci 5:146–156 5. Morgenstern DA, Asher RA, Fawcett JW (2002) Chondroitin sulphate proteoglycans in the CNS injury response. Prog Brain Res 137:313–332 6. Schwab ME, Bartholdi D (1996) Degeneration and regeneration of axons in the lesioned spinal cord. Physiol Rev 76:319–370 7. Carulli D, Laabs T, Geller HM et al (2005) Chondroitin sulfate proteoglycans in neural development and regeneration. Curr Opin Neurobiol 15(2):252 8. Yamashita T, Higuchi H, Tohyama M (2002) The p75 receptor transduces the signal from myelin-associated glycoprotein to Rho. J Cell Biol 157(4):565–570 9. Mi S, Lee X, Shao Z et al (2004) LINGO-1 is a component of the Nogo-66 receptor/p75 signaling complex. Nat Neurosci 7:221–228 10. Yamashita T, Fujitani M, Hata K et al (2005) Rho/Rho-kinase as potential therapeutic target for CNS injury. Gene Ther Mol Biol 9:265–268 11. Caroni P, Schwab ME (1988) Antibody against myelin-associated inhibitor of neurite growth neutralizes nonpermissive substrate properties of CNS white matter. Neuron 1:85–96 12. Grandpre´ T, Nakamura F, Vartanian T et al (2000) Identification of the Nogo inhibitor of axon regeneration as a Reticulon protein. Nature 403(6768):439–444 13. Huber AB, Weinmann O, Bro¨samle C et al (2002) Patterns of Nogo mRNA and protein expression in the developing and adult rat and after CNS lesions. J Neurosci 22(9):3553–3567 14. Prinjha RK, McAdam RA, Burbridge SA et al (2004) Strategies for developing Nogo antagonists. J Drug Discov Today Ther Strateg 9:21–27
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15. Prinjha R, Moore SE, Vinson M et al (2000) Inhibitor of neurite outhgrowth in humans. Nature 403:383–384 16. Oertle T, Klinger M, Stuermer CAO et al (2003) A reticular rhapsody: phylogenic evolution and nomenclature of the RTN/ Nogo gene family. FASEB J 17(10):1238–1247 17. McKerraccher L, David S, Jackson DL et al (1994) Identification of myelin-associated glycoprotein as a major myelin-derived inhibitor on neurite growth. Neuron 13(4):805–811 18. Cad D, Qiu J, Cao Z et al (2001) Neuronal cyclic AMP controls the developmental loss in ability of axons to regenerate. J Neurosci 21(13):4731–4739 19. Huang JK, Phillips GR, Roth AD et al (2005) Glial membranes at the node of Ranvier prevent neurite outgrowth. Science 310:1813–1817 20. Atwak JK, Pinknston-Gosse J, Syken J et al (2008) PirB is a functional receptor for myelin inhibitors of axonal regeneration. Science 322:967–970 21. Wong ST, Henley JR, Kanning KC et al (2003) A p75 (NTR) and Nogo receptor complex mediates repulsive signaling by myelinassociated glycoprotein. Nat Neurosci 5(12):1302–1308 22. Fournier AE et al (2001) Identification of a receptor mediating Nogo-66 inhibition of axonal regeneration. Nature 18(409): 341–346 23. Grandpre´ T, Li S, Strittmatter SM (2002) Nogo-66 receptor antagonist peptide promotes axonal regeneration. Nature 417(6888):547–551 24. Li S, Strittmatter SM (2003) Delayed systemic Nogo-66 receptor antagonist promotes recovery from spinal cord injury. J Neurosci 23(10):4219–4227 _ ´ A et al (2008) Rodzina białek Rho 25. Nowak JM, Grzanka A, Zuryn i ich rola w cytoszkielecie komo´rki. Postepy Hig Med Dosw 62:110–117 26. Saski T, Takai Y (1998) The Rho small G protein family-Rho GDI system as a temporal and spatial determinant for cytoskeletal control. Biochem Biophys Res Commun 245:641–645 27. Dergham P, Ellezam B, Essagian CH et al (2002) Rho signaling pathway targeted to promote spinal cord repair. J Neurosci 22(15):6570–6577 28. Fournier A, Takizawa B, Strittmatter SM et al (2003) Rho kinase inhibition enhances axonal regeneration in the injured CNS. J Neuosci 23(4):1416–1423 29. Yamashita T, Tohyama M (2003) The p75 receptor acts as a displacement factor that releases Rho from Rho-GDI. Nat Neuosci 6L:461–467 30. He Z, Koprivica V (2004) The Nogo signaling pathway for regeneration block. Annu Rev Neuosci 27:341–368 31. Bandtlow CE, Schmidt MF, Hassinger TD et al (1993) Role of intracellular calcium in NI-35-evoked collapse of neuronal growth cones. Science 259:80–83 32. No´gra´di A, Szabo´ A, Pinte´r S et al (2007) Delayed riluzole treatment is able to rescue injured rat spinal motoneurons. Neuroscience 144:431–438
REVIEW ARTICLE
Axonal regeneration inhibitors: emerging therapeutic options T. W. Rosochowicz • S. Wrotek • W. Kozak
Received: 22 September 2014 / Accepted: 30 December 2014 Ó Belgian Neurological Society 2015
Abstract For the most part, the central nervous system is unable to regenerate. The majority of injuries of vascular, inflammatory, degenerative and traumatic aetiology lead to an irreversible loss of central nervous system function. The paper presents the role of Nogo-A, MAG and OMgp proteins in the inhibition of central nervous system regeneration, and their potential clinical significance. Keywords Nogo-A MAG OMgp Regeneration of the central nervous system
takes some time. Some proteins, including Nogo, myelinassociated glyocoprotein (MAG) and oligodendrocyte myelin glycoprotein (OMgp), are released at the moment of injury of the oligodendrocytes and myelin sheaths. Those proteins demonstrate strong axonal regeneration inhibiting properties. It is believed that they play a major role in the inhibition of regeneration of the CNS. The purpose of this paper is to provide a brief presentation of the myelinassociated proteins that inhibit regeneration of axons, as well as some of their inhibitors, applied in preliminary animal models and in first human clinical trials.
Introduction Axon regeneration scheme In addition to some structures of the hypothalamus and neurons localised within the olfactory bulb, the central nervous system (CNS) is depleted of regenerative properties. Therefore, obvious and significant regeneration of cerebral and spinal neurons that could restore the function lost in the course of various neurological conditions, including cerebral strokes, cerebro-cranial injuries, neuroinfections, sclerosis multiplex and broadly understood neurodegenerative diseases, has not been observed. In contrast to the CNS, the peripheral nervous system presents some significant regenerative properties. Injury of the CNS is associated with damage to neurons and glial cells. Depending on the aetiological factor, the injury may be of mechanical, ischaemic, immunological or inflammatory origin. A glial scar is gradually formed, constituting a physical barrier and containing substances that inhibit regeneration of neurons. However, glial scar formation T. W. Rosochowicz (&) S. Wrotek W. Kozak Nicolaus Copernicus University, Torun´, Poland e-mail: [email protected]
It was experimentally demonstrated that, following an injury, regenerative growth cones appear on the ends of axons [1]. Ultrastructural evaluation revealed the presence of lamellar structures, referred to as lamellipodia, possessing smaller outgrowths, known as filopodia. When axon continuity is compromised in favourable conditions, i.e., in presence of Schwann cells, it creates a necessary scaffolding allowing regeneration of the axon. That phenomenon was already demonstrated in experimental studies at the beginning of the 20th century [2]. If stimulating factors are present, the axon begins to grow. In the peripheral nervous system, complete or partial axon function restoration occurs following an injury, depending on its type (e.g., according to Seddon-Sunderland classifications of 1943 and 1957). The central nervous system has no regenerative properties, because of the presence of inhibitors of the axonal regeneration. At the moment of injury, those inhibitors are released from myelin. Similar properties are also demonstrated by a newly forming glial scar. In early 1989, an experiment was conducted [3] demonstrating that non-
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myelinised nerve fibres present in the CNS have the ability to regenerate following a chemical axonotomy. However, the process does not occur in the neighbourhood of the myelinised fibres. The presence of myelin leads to the release of regeneration inhibiting factors, resulting in decay of the fibres.
(GTP-ase) RhoA activates a Rho-dependent kinase (ROCK) [10]. That activation leads to increased rigidity of the actin cytoskeleton resulting in inhibition of the axonal growth cone and in its further elongation. Calcium ions are another secondary messenger. An increase in their intracellular level leads to reduced cAMP concentration, and that phenomenon causes inhibition of axonal regeneration (see Fig. 1).
The glial scar Within approximately 14 days following an injury to the central nervous system, a glial scar is formed preventing axons from growing [4]. The scar is formed of fibroblasts originating from meningeal cells, astrocytes, oligodendrocytes and microglia. Those cells aggregate at the site of an injury. The process has two implications: it creates a physical barrier hindering penetration of budding axons or even preventing the penetration completely, and the forming layer constitutes a source of substances, such as of proteoglycans chondroitin sulphate and keratan sulphate, which inhibit axonal regeneration [5]. Those compounds possess some very strong inhibiting properties that impact nervous system regeneration. In addition, a major role is played by the myelin-associated axonal regeneration inhibitors described below [6]. Proteoglycans are huge molecules abundant in the human organism. They are composed of a protein core covalently bound to chains of glucosaminoglycans, e.g., heparan sulphate. The enzymechondroitinase [7] separates those chains, thereby depleting the axon regeneration inhibiting properties of those compounds.
Myelin-associated inhibitors of axonal regeneration Myelin contains three important proteins responsible for inhibition of axonal regeneration: Nogo-A, MAG and OMgp, previously known as arretin. All three of these proteins have a common NgR1 receptor (NgR-Nogo receptor). MAG also binds to a second subtype of that receptor: NgR2. The NgR1 receptor is a GPI-anchored protein. Glycosylphosphatidylinositol (GPI) is a glycolipid allowing protein anchoring in so-called lipid rafts that binds to a protein C-terminal. Formation of the complex is necessary for activation of the receptor. The literature reports other subunits of that complex: p75NTR (low affinity neurotrophin receptor) [8], TROY (sometimes referred to as TAJ) and LINGO-1 (leucine-rich repeat and immunoglobulin-like domain-containing Nogo receptorinteracting protein) [9]. A signal may be transmitted further only after the complex is formed. There are two secondary messengers: RhoA and an increase in the concentration of intracellular calcium ions. Guanozino-50 triphosphatase
123
Nogo An anti-myelin monoclonal antibody IN-1 was successfully created in 1988 [11]. The antibody was produced in order to block the axonal regeneration inhibiting properties of the myelin. Later it was demonstrated that the IN-1 monoclonal antibody is targeted at Nogo-A [11]. IgM class antibodies were used in the 1970s, and more recent studies employ IgG class antibodies. Several years later, in 2000 [12], the Nogo gene was successfully cloned, and its principal product, Nogo-A, was demonstrated to be the proper antigen for the IN-1 antibody. Nogo is one of the most important and the best-studied proteins responsible for the inhibition of axon growth in an injured central nervous system. In the nervous system, Nogo-A is a membranous protein present principally in oligodendrocytes and myelin sheaths. Moreover, the Nogo family includes several isoforms, Nogo-B and Nogo-C, which play no significant role in regeneration of the nervous system. Nogo-B is present in various tissues and Nogo-C is principally encountered in muscular tissue [13]. Each of those three proteins is coded by the same Nogo gene. They all share the same C-terminal chain of 188 amino-acids. Due to that amino-acid sequence Nogo proteins may be included in a small family of reticulon proteins (Nogo-A is also referred to as RTN4), present in the nervous system. Nogo proteins are characterised by the presence of two hydrophobic sections connected with a loop consisting of 66 amino-acids. Studies have indicated that those hydrophobic sections demonstrated particular axon regeneration inhibiting properties. Therefore, the sequence is referred to as ‘‘Nogo-66’’ [14]. It is present in all isoforms of the Nogo protein. It demonstrates a high affinity to the NgR receptor. The Nogo-A isoform, and its specific N-terminus, is recognised by the monoclonal antibody IN-1 [15]. It is notable that the Nogo-A protein is absent in some animals, including salamanders [16] and fish that possess some astounding regenerative properties, indicating that it is involved in regeneration of some body parts (such as tails) and, therefore, also of the spinal cord. Thus, there is important evolutionary evidence for the inhibitory effect of the Nogo-A protein on axonal regeneration in the central nervous system (see Fig. 2).
Acta Neurol Belg Fig. 1 Action mechanisms of myelin-associated inhibitors of axonal regeneration on receptors (according to Schwab 2005)
Fig. 2 Nogo protein isoforms
Myelin-associated glycoprotein (MAG) Myelin-associated glycoprotein (MAG) is one the first proteins discovered to be able to inhibit axonal regeneration [17]. MAG is present in the periaxonal space and plays
a significant role in mutual myelin-axon interaction. For example, it is associated with the onset of myelinisation, maintenance of myelin and myelin sheath integrity. MAG has double properties; it is an axonal growth factor but it also inhibits axonal regeneration. MAG has an effect on
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neuron changes during ontogenesis; during the foetal period, it is a stimulant and when myelinisation is complete, MAG changes into a suppressant [18].
receptor type B (LILRB2). The NgR receptor is associated with complex-forming subunits: p75, LINGO-1 and TROY [21]. As previously mentioned, the Rho-A/ROCK system is an effector for the NgR complex (see Fig. 3).
Oligodendrocyte myelin glycoprotein (OMgp) Clinical significance As indicated by its name, OMgp is localised principally in oligodendrocytes, but it is also encountered in neurons. Via glycophosphatydylinositol it is anchored in the plasmic membrane. This protein inhibits axonal regeneration [19].
Receptors of axonal regeneration inhibitors The NgR receptor (proper name NgR1) is a glycoprotein anchored in the plasmic membrane with glycophosphatydylinositol. The receptor demonstrates affinity towards Nogo-A, MAG and OMgp. The NgR2 isoform of the receptor also binds to MAG. Another recently discovered receptor, paired immunoglobulin-like receptor type B (PirB) [20], also shows affinity to all the myelin-associated proteins mentioned above, thereby inhibiting axonal regeneration. Even blocking the NgR receptor could not restore the regenerative properties of axons located in the central nervous system. That phenomenon is most probably associated with the presence of the other receptor, PirB. That receptor was discovered in animal studies. In humans, a similar role is played by leukocytic immunoglobulin-like
Fig. 3 The NgR1 receptor activation mechanism (according to Oertle 2003)
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Nogo extracellular peptide, residues 1-40 (NEP 1-40) is a competitive agonist of the NgR receptor [22]. In experimental injury of the spinal cord, intrathecal administration of NEP1-40 [23] clearly improved regeneration of axons in the spinal cord. Of course, use of the peptide in acute spinal cord injury may be difficult. Considering that fact, Li [24] studied subcutaneous administration of the NEP 1-40 peptide. Even administration of the study substance a week after an injury proved not to be inferior compared to administration of the substance at the moment of the spinal cord injury. The study demonstrated an intensified axonal growth in the cortical-medullary pathways and ‘‘budding’’ of serotoninergic fibres, as well as reconstruction of synapses. Improvement of motor function of paralysed muscular groups was also achieved. The Rho protein family [25] is characterised by the presence of guanosine-50 triphosphatase (GTP-ase)-containing domains. Due to their presence, the proteins are referred to as molecular switches. Each of those proteins may be in the active GTP (guanosine-50 triphosphate)-containing form or the inactive GDP (guanosine-50 diphosphate)-containing
Acta Neurol Belg Fig. 4 Mechanism of action of selected compounds restoring axonal regeneration
form. The p75NTR subunit releases Rho from the Rho-GDI (guanine nucleotide dissociation inhibitor) complex and inhibits transition from the inactive to the active form [26]. ROCK is another protein activated by Rho-A. Considering that fact, it was assumed that inhibition of the Rho-A/ROCK system may lead to secondary improvement of axonal regeneration in the CNS. The Rho kinase inhibitor known as Y-27632 may be an example of a potential pharmacological block of the Rho-A/ROCK system. Its mechanism of action consists in competitive binding to the kinase at the ATPbinding site. A similar role is played by a bacterial (obtained from Clostridium botulinum) endotoxin transferase C3, which is also able to inhibit Rho via the process of ADP ribosylation. In a comparative study, Dergham [27] demonstrated the superior efficiency of C3 transferase compared to Y-27632. Both proteins also demonstrate neuroprotective properties. Fournier’s experiments [28] on an animal model demonstrated that application of Y-27632 enabled and improved regeneration of cortical-medullary pathways, and speeded up the return of the motor activity. The C3 transferase proved to be less efficient in that study. A short Pep5 peptide is another compound that is potentially able to support regeneration of the nervous system is. The peptide binds at the site of connection between p75NTR and Rho-GDI. Its efficacy was demonstrated as it allowed regeneration of neurons in spinal ganglions and in the cerebellum [29]. In the US, clinical trials are currently being carried out with the C3 transferase
analogue BA-210 (Cethrin), administered intrathecally to patients with acute cervical and thoracic spine injury. Rolipram—a phosphodiesterase 4 inhibitor—is another promising medicinal product. The drug is used principally to treat depressive syndrome, and it demonstrates antipsychotic properties as well as anti-inflammatory properties. It was also demonstrated that, by increasing the intracellular cAMP level, the drug improved the regenerative properties of neurons [30]. Dantrolene is a compound that inhibits intracellular calcium ion release. Administration of the drug blocks Ca2?, thus suppressing NI-35 (neuron growth inhibitor). That prevents degeneration of a growth cone [31]. Experimental studies demonstrated that Riluzol, approved for treating SLA, may also be applicable to spinal cord injuries, improving re-innervation even if administered several days after the injury [32] (see Fig. 4).
Conclusion A brief description of the proteins that inhibit axonal regeneration, the role of glial scars and of the Rho-A/ ROCK system presented in this paper indicate that potential possibilities exist for reversing of the currently ‘‘irreversible’’ damage of the CNS. Although only selected studies have been presented, the majority of them were completed on animal models. However, as the first clinical
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trials with humans emerge, using classical methods of neurological therapy and modern neurosurgical procedures, the process of ‘‘irreversible’’ injury of the CNS will soon be successfully reversed. Acknowledgments
This study received no financial support.
Conflict of interest The authors report no financial or other conflict of interest associated with this study.
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