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Immunization with myelin or recombinant Nogo-66/MAG in alum promotes axon regeneration and sprouting after corticospinal tract lesions in the spinal cord

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Abstract

We have shown previously that immunization with myelin in incomplete Freund’s adjuvant (IFA) is able to promote robust regeneration of corticospinal tract fibers in adult mice. In the present study the effectiveness of such immunization with myelin was compared to that of a combination of two axon growth inhibitors in myelin, Nogo-66 (the 66-amino-acid inhibitory region of Nogo-A) and myelin-associated glycoprotein (MAG). The effectiveness of two adjuvants, IFA and aluminum hydroxide (Alum), was also compared, the latter being one that can be used in humans. In addition, larger dorsal overhemisections were made at the lower thoracic level, which resulted in a larger scar. These studies were carried out in SJL/J mice, a mouse strain that is susceptible to autoimmune experimental allergic encephalomyelitis (EAE). None of the immunized mice developed EAE. Long-distance axon regeneration and sprouting of the corticospinal tract was seen in myelin and Nogo-66/MAG immunized mice. Alum was as effective or better than IFA as the adjuvant. Overall, the robustness of axon growth and sprouting was greater in mice immunized with myelin. The abundance of this growth was less than in our earlier work in which smaller lesions were made, pointing to the possible influence of inhibitors in the scar. This work shows, however, that axon growth inhibitors in myelin can be selectively blocked using this immunization approach to promote long-distance axon regeneration in the spinal cord.

Introduction

The failure of damaged axons to regenerate for long distances to reconnect with their targets contributes to the motor and sensory deficits seen after spinal cord injuries in adult mammals. Although multiple factors are likely to underlie the failure of axon regeneration, axon growth inhibitors associated with myelin (Bandtlow and Schwab, 2000) and the glial scar Davies et al 1997, Davies et al 1999, Moon et al 2001, Bradbury et al 2002 appear to play an important role. Myelin has several neurite growth inhibitory activities McKerracher et al 1994, Li et al 1996, three of which have been identified as Nogo-A (Caroni and Schwab, 1988), myelin-associated glycoprotein (MAG) Mukhopadhyay et al 1994, McKerracher et al 1994, and oligodendrocyte-myelin glycoprotein (OMgp) (Wang et al., 2002). Nogo-A inhibits neurite growth in vitro and induces growth cone collapse Chen et al 2000, GrandPré et al 2000, Prinjha et al 2000. The IN-1 monoclonal antibody directed against Nogo-A is able to promote regeneration and sprouting of axons after CNS injury in adult mammals Schnell and Schwab 1990, Fouad et al 2001, Raineteau et al 2001. Two neurite growth inhibitory domains in Nogo-A have been identified Chen et al 2000, GrandPré et al 2000. One of these domains is a 66-amino-acid region (Nogo-66) that forms an extracellular loop GrandPré et al 2000, Chen et al 2000, Wang et al 2002. The other inhibitory domain of Nogo-A is located near the N-terminal region (Amino Nogo) that inhibits neurite growth and fibroblast spreading Chen et al 2000, GrandPré et al 2000, Liu et al 2002.

A number of studies have demonstrated that MAG has potent neurite growth inhibitory activity in vitro Mukhopadhyay et al 1994, McKerracher et al 1994, De Bellard et al 1996, David et al 1999. MAG also induces growth cone collapse specifically of axonal but not dendritic growth cones (Shibata et al., 1998). The neurite growth inhibition induced by myelin and MAG in vitro is mediated by activation of RhoA, a member of the Rho GTPase family Lehmann et al 1999, Dergham et al 2002. Increasing cAMP levels or inactivating RhoA leads to improved regeneration after spinal cord injury Qui et al 2002, Neumann et al 2002, Dergham et al 2002. Recently, OMgp, a glycosylphosphatidylinositol-anchored protein, was reported to also have potent growth cone collapsing and neurite growth inhibitory properties (Wang et al., 2002). All three myelin-associated inhibitors, the Nogo-66 region of Nogo-A, MAG, and OMpg, bind to the same high-affinity receptor, first identified as the Nogo receptor Fournier et al 2001, Wang et al 2002, Liu et al 2002, Domeniconi et al 2002. Blocking this receptor with a peptide results in improved axon regeneration and sprouting after corticospinal tract lesions in adult rats (GrandPré et al., 2002).

We have previously shown that immunizing mice with purified myelin in incomplete Freund’s adjuvant (IFA) results in the generation of anti-myelin antibodies that are able to neutralize the inhibitory effects of myelin substrates in vitro (Huang et al., 1999). Such immunizations also resulted in robust regeneration of damaged axons of the corticospinal tract in adult mice (Huang et al., 1999). In these experiments, IFA was used as the adjuvant because it is known to prevent experimental allergic encephalomyelitis (EAE) and results in antibody production Rodriguez et al 1987, Rivero et al 1997. In addition, to minimize the contribution of the glial scar, small dorsal hemisections were made to sever the corticospinal tract with lesions extending only up to the central canal. In the present work we have carried out experiments in which larger dorsal overhemisections were made that extended well beyond the central canal. In these experiments we compared the effects of immunization with myelin with that of a cocktail of the two known inhibitors, Nogo-66 and MAG, and also compared the efficacy of two adjuvants, IFA and aluminum hydroxide (Alum).

Section snippets

Results

In these experiments two parameters were assessed: (1) the effectiveness of immunization with myelin as compared to that of a cocktail of rNogo-66/rMAG; and (2) the effectiveness of two adjuvants, IFA and Alum. None of the immunized mice developed EAE. Mice were evaluated based on a 5-point clinical score that is routinely used in the assessment of EAE in mice. Mice with EAE get a paralytic attack within the first 7–10 days after immunization. The 5-point clinical scale for evaluating EAE mice

Discussion

In this study, the SJL/J mouse strain which is susceptible to EAE Bernard 1976, Constantinescu et al 2001 was used in an effort to obtain evidence that the immunization protocols tested would not trigger an autoimmune disease against myelin or the myelin antigens tested. Generally, SJL/J mice immunized with complete Freund’s adjuvant and myelin or certain myelin antigens show signs of EAE within 7–10 days. None of the animals immunized with myelin or rNogo-66/rMAG in IFA or Alum developed any

Preparation of myelin

Bovine CNS myelin was prepared using a previously described protocol (McKerracher et al., 1994). Briefly, CNS white matter from bovine brain was homogenized with a Dounce homogenizer in 0.32 M sucrose on ice. The homogenate was overlaid on 0.85 M sucrose and centrifuged for 30 min at 75,000g at 4°C. The interface was collected, resuspended in ice-cold water, and centrifuged for 20 min at 25,000g. The pellet was again resuspended in ice-cold water and centrifuged for 10 min at 10,000g. The

Acknowledgements

This work was funded by a grant from the Canadian Institute of Health Research (CIHR). M.S. was the recipient of a studentship from the MS Society of Canada.

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