The “intrinsic hypothesis” is challenged by the fact that PNS neurons exhibit throughout their life a strong regenerative ability (Schwab & Thoenen 1985). Furthermore, neurons such as those in dorsal root ganglia (DRG) have axons in both the CNS and PNS, but can only regenerate their peripheral processes. These observations strongly suggest that it is in fact CNS factor(s) of an external nature which inhibit(s) the nerve regeneration (Qiu et al. 2000, Yiu & He 2006). In 1981 this new paradigm (even if Cajal, here again, had already suggested this in 1905) was empirically demonstrated by David and Aguayo who emphasized that the axon growth of CNS lesioned neurons is increased if they were provided with a peripheral environment which is radically different (e.g.
Schwann cells instead of oligodendrocytes) from the CNS (David & Aguayo 1981). As a consequence concept of “glial inhibition” was born.
From now on it appears that the post-natal development of CNS glial cells and their degeneration (i.e. the myelinic debris) coupled to their reactions consecutively to a given insult are responsible for the progressive decline of axon regrowth. In other words, the neurons do not lack an intrinsic ability to regenerate. This first decisive step will be followed in the early 1990’s by the discovery of the first inhibitory molecules, their receptors as well as their signaling.
The Oligodendrocytes/myelinic inhibitor, Nogo
In the late 1980’s Martin Schwab and colleagues, from the University of Zurich, isolated a monoclonal antibody that neutralize the action of a fraction of myelin, termed IN-1, which was previously shown to inhibit neurite extension in vitro (Schnell & Schwab 1990, Caroni & Schwab 1988). Moreover when the antibody against IN-1 was injected into adult rat spinal cord injury, about 5% of the severed axons regenerated across the injured tissue and the animals showed striking functional recovery of their motor functions (Bregman et al. 1995). These spectacular data obtained with this antibody became definitely revolutionary when they were also confirmed in adult monkeys that recovered a complete manual dexterity after cervical lesion (Freund et al. 2006).
Naturally these results obtained in a pre-clinical test (carried-out by Novartis and Martin Schwab) have raised high hopes in this strategy of therapy applied to CNS injuries in human. Consequently a phase I clinical trial of intrathecal infusion of anti-IN1 is being carried-out in Europe and one other will begin in North America (Ruff et al. 2008).
Later the antigen for the IN-1 antibody was cloned and was named Nogo (in reference to the
McMahon 2006, Yiu & He 2006, Filbin 2003). Nogo has been shown to belong to the reticulon family and to exist in three distinct forms (Nogo-A,-B and -C) that are generated by alternative splicing (Figure 14). All three Nogo have a common carboxyl (C) terminus. Among these, Nogo-A is the best characterized due to its high expression in oligodendrocytes and myelin (Huber et al.
2002). Structure-function analysis revealed that two inhibitory domains are present in NogoA: a unique amino (N) terminus (amino Nogo) and a 66 amino acids loop (Nogo-66) that is common to all isoforms (Prinjha et al. 2000, Chen et al. 2000).
Figure14: Structure of the three Nogo isoform.
All the three isoforms have a common inhibitory domain, Nogo66. Among them, Nogo-A is the only to possess a second inhibitory domain, Amino-Nogo.
The Nogo receptors
In the early 2000’s using a soluble form of Nogo-66, Strittmatter and colleagues cloned a binding partner for Nogo-66 which they called…Nogo receptor (NgR) (Fournier et al. 2001). This latter is a GPI-linked protein and was found to be expressed on the surface on main neuronal types of the CNS (Josephson et al. 2002). Remarkably, NgR has been shown to interact with high affinity to MAG (Domeniconi et al. 2002) and OMgp (Wang et al. 2002b), which are also myelinic inhibitors of the regeneration, but nonetheless structurally different from Nogo suggesting a common pathway for the inhibition of the regeneration.
However NgR lacks an intracellular domain and by consequence cannot transduce the myelin- associated signal suggesting the binding of this complex with a co-receptor. Later, p75, a member of the tumour necrosis factor receptor family, was revealed to be the co-receptor of Nogo. Indeed p75 mutant do not respond to Nogo-66 as well as the other myelinic inhibitors (Yamashita et al.
2002, Wang et al. 2002a). In addtion p75 form a physical receptor complex with NgR (Wong et al.
2002, Wang et al. 2002a).
The Rho kinase (ROCK) pathway
The identification of the complex NgR/p75 involves that a specific signal is initiated when the growth cone of neurons are in contact with the inhibitory signal rather than a myelin-derived non permissiveness.
McKerracher’s group, from the University of Montreal, was the first to identify the Rho family of small GTPase proteins (see figure 13) to be involved in mediating the inhibitory effect of Nogo and this before the discovery of the NgR/p75complex (Lehmann et al. 1999). This is not surprising since as mentioned previously (see pages 29 to 31) these GTPases of the Rho family are known to be regulator of actin cytoskeleton (Hall 1998). In their study they treated injured primary retinal neurons with C3 enzyme (which inactivates Rho) and observed a neuritic extension on inhibitory substrates (Figure 15). Furthermore, the involvement of Rho in mediating the inhibitory signal of the myelin has been clearly established in vivo since application of C3 to the site of spinal cord injury results in extensive regeneration and functional recovery in mice (Dergham et al. 2002). The downstream effector of Rho which is involved in mediating the myelin inhibition was revealed to be the Rho kinase (ROCK) since its inactivation overcomes the Nogo/MAG-mediated inhibition (Fournier et al. 2003). The main further downstream effectors (Dickson 2001, Sandvig et al. 2004, Hsieh et al. 2006) that have been highlighted are the LIM (Lin-11, IsL-1, Mec-3) kinases Slingshot phosphatase (see figures 15) which regulate the actin depolymerization factor Cofilin (in both cases, the final outcome is to prevent the actin recycling toward the leading edge of the growth cone which finally collapses).
Figure 15: Signaling of the myelin-associated inhibitor. (adapted from Filbin 2003)
The glial scar and the chondroitin sulfate Proteoglycans
In addition to degenerating myelin another important source of inhibition of axonal growth is the formation of the glial scar or astrogliosis. This glial reaction involved mainly the recruitment of microglia and astrocytes to the lesion site. Some of these reactions are recognized to exert beneficial effects like the isolation of the injury site or even more the support of axon regrowth. However most of the astrocytes become hypertrophic and adopt a reactive phenotype. In the same time they increase their expression of extracellular matrix molecules, the chondroitin sulfate Proteoglycans (CSPGs) which have been extensively shown to inhibit axonal regrowth (Davies et al. 1997). The CSPGs (aggrecan, brevican, neurocan, versican, phospacan and NG2) are a family of molecules characterized by a core protein to which large and highly sulphated glycosaminoglycan (GAG) are attached (Morgenstern et al. 2002). After an injury the CSPG expression is rapidly upregulated forming an inhibitory gradient that is highest at the centre of the lesion (see Figure 16). It has been clearly shown that it is the GAG rather than the core itself that exerts an inhibitory activity on
lesioned axons. Indeed a treatment with the bacterial enzyme, the chondroitinase ABC, abolishes the inhibition (Silver & Miller 2004).
To date the mechanisms by which the CSPG exert effect on axonal regrowth are still not clear.
Various studies suggest that they act as a mechanical barrier masking neurons from neurite promoting components of the extracellular matrix such as the laminin and N-CAM molecule (Bovolenta & Fernaud-Espinosa 2000).
Figure 16: Dystrophic growth cone are submitted to an hostile environment
Injury to the adult CNS leads often to a transection of transection of fibers and damages glial cells such as oligodendrocytes and myelin debris. The dystrophic growth cones are then exposed to myelin/oligodendrocytes inhibitors as well as reactive astrocytes that form a glial scar and produce CSPGs which increase the blockade of axonal growth.
To summarize, throughout the three last decades major advances have permitted to establish a new paradigm in regard to what are the drive of the CNS regeneration. Hence the concept of “glial inhibition” has mainly permitted to highlight that the progressive decline in the ability to regenerate was mainly due to the environment to which the dystrophic growth cone are exposed (figure16).
This has brought to new approaches consisting of blocking one of several glial inhibitors and/or
their receptor or more judiciously, because they share a common co-receptor (p75), one of their downstream effectors (Schwab et al. 2006, Yiu & He 2006).
However one mirror but complementary other approach consists to change the intrinsic state of the neurons so that they overcome the environmental inhibitors.