Elsevier

Neurobiology of Disease

Volume 51, March 2013, Pages 202-213
Neurobiology of Disease

Long-distance axonal regeneration induced by CNTF gene transfer is impaired by axonal misguidance in the injured adult optic nerve

https://doi.org/10.1016/j.nbd.2012.11.011Get rights and content

Abstract

The optic nerve crush injury is a well-accepted model to study the mechanisms of axonal regeneration after trauma in the CNS. The infection of retinal ganglion cells (RGCs) with an adeno-associated virus serotype 2 — ciliary neurotrophic factor (AAV2.CNTF) was previously shown to stimulate axonal regeneration. However, the transfection of axotomized neurons themselves may not be optimal to promote full axonal regeneration in the visual system. Here, we show that the release of CNTF by glial cells is a very powerful stimulus for optic fiber regeneration and RGC survival after optic nerve crush. After 8 weeks, long-distance regeneration of severed optic axons was induced by CNTF until and beyond the optic chiasm. Regenerated axons stayed for at least 6 months in the damaged optic nerve. Strikingly, however, many regenerated axons showed one or several sharp U-turns along their course, suggesting that guidance cues are missing and that long-distance axonal regeneration is limited by the return of the growing axons toward the retina. Even more surprisingly, massive axonal sprouting was observed within the eye, forming a dense plexus of neurites at the inner surface of the retina. These results indicate that massive stimulation of the neuronal growth program can lead to aberrant growth; the absence of local regulatory and guidance factors in the adult, injured optic nerve may therefore represent a major, so far underestimated obstacle to successful axon regeneration.

Highlights

► A new gene therapy approach is proposed to protect injured retinal neurons using a glia-targeting AAV vector. ► A CNTFRα super-agonist was a potent activator of lesioned retinal ganglion cell survival. ► Long-distance axonal regeneration in the injured optic nerve was sustained by CNTFRα super-agonist delivery. ► The treatment with a modified CNTF peptide did not exacerbate gliosis after lesion. ► Axonal misguidance is a major limitation to long-distance axonal regeneration in the adult visual system.

Introduction

The optic nerve crush model was extensively used to study the mechanisms of axonal growth inhibition and to design new repair strategies for the injured CNS (Benowitz and Yin, 2007, Harvey et al., 2006). In the intraorbital optic nerve crush paradigm, severed axons cannot spontaneously regenerate into the distal part of the optic nerve and most of the retinal ganglion cell (RGC) bodies die by apoptosis after 2 weeks (Berkelaar et al., 1994).

Contrary to BDNF (Mansour-Robaey et al., 1994) or FGF2 (Sapieha et al., 2003), CNTF was shown to stimulate both axonal regeneration and neuronal survival after optic nerve lesion (Lingor et al., 2008, Muller et al., 2007, Muller et al., 2009). Repeated intraocular injections of the recombinant CNTF peptide were efficient at activating axonal growth and neuronal survival but only to a limited extent (Muller et al., 2007). The effects of CNTF are likely restricted in time by the short half life of the recombinant peptide (Dittrich et al., 1994) and by the negative feedback control mediated by the up-regulation of the suppressor of cytokine signaling 3 (SOCS3) (Smith et al., 2009). To sustain the CNTF delivery in the retina, an adeno-associated virus serotype 2 (AAV2) containing the Cntf cDNA was intravitreally injected to selectively infect the RGCs. AAV2.CNTF treatment resulted in significant neuroprotection and regeneration of some optic axons over longer distances (Leaver et al., 2006a, Leaver et al., 2006b). However, transducing neurons may not be optimal to deliver survival factors to the retina as only a small number of cells was infected (Leaver et al., 2006b) and protein synthesis is altered in axotomized neurons (Park et al., 2008).

Here, we hypothesized that the Müller glia-mediated release of CNTF may improve neuroprotection and stimulate long-distance axonal regeneration. In the healthy retina, Müller cells fulfill similar homeostatic functions as astrocytes in the rest of the CNS (Bringmann et al., 2006). Müller cell bodies occupy a central position in the retina from where they extend radial processes contacting all types of retinal neurons. In the degenerating retina, Müller cells are resistant to cell death and therefore are ideal intermediates to release neurotrophic factors. After optic nerve lesion, the Müller cell response is characterized by strong reactive gliosis and by a small number of proliferating cells (Wohl et al., 2009). AAVs allow stable, safe and efficient gene transfer and are thus suitable for human gene therapy (Bainbridge et al., 2008, Maguire et al., 2008, Maguire et al., 2009). An engineered AAV called ShH10 was selected based on its ability to preferentially transduce Müller glia (Klimczak et al., 2009). Here we present the effects of the infection of Müller cells by the ShH10 vector carrying the cDNA of DH-CNTF, a mutant peptide exhibiting a higher affinity for CNTFRα and therefore acting as a super-agonist for this receptor subunit (Saggio et al., 1995). Our results show that glia-targeting AAV.DH-CNTF can promote long-range axonal regeneration. However, the distance covered by the regrowing axons was severely limited by the frequent formation of U-turns in the optic nerve. In addition, we observed massive aberrant axonal sprouting at the inner surface of the retina. Our data suggest that axonal misguidance is a key limiting factor for the long-distance axonal regeneration in the visual system.

Section snippets

Animals

All surgeries were performed on 2–4 month old male C57BL/6 mice. Animal experiments were performed in agreement with the guidelines of the Veterinary Office of the Canton of Zürich.

ShH10 vector production

The AAV transfer plasmid with a modified form of the ciliary neurotrophic factor gene, DH-CNTF (Fig. 1C) was a generous gift from Dr W. Hauswirth, University of Florida. DH-CNTF recognizes with higher affinity the CNTFRα (Saggio et al., 1995) subunit (Fig. 1C). This construct contained a growth hormone signal peptide

ShH10.DH-CNTF selectively infects Müller glia and activates the Jak3/Stat3 pathway in retinal ganglion cells

Four weeks after ShH10.GFP delivery, most of the GFP expressing cell bodies were localized in the middle of the inner nuclear layer and could be stained for glutamine synthase, a marker for the Müller cells (Fig. 1A) (Dalkara et al., 2011, Klimczak et al., 2009). At this time point, ~ 84% of Müller cells expressed the GFP protein. Eight weeks following optic nerve crush (12 weeks post ShH10.GFP injection), a very high density of GFP-positive Müller cells was visualized by confocal microscopy on

Discussion

By selectively and efficiently infecting the retinal glial cells with a new AAV variant, we could deliver the CNTFRα super-agonist DH-CNTF to RGCs thereby causing a long-lasting and potent activation of the Jak3/Stat3 pathway, a key regulator of neuronal growth. ShH10.DH-CNTF induced long-distance, sustained axonal regeneration through the crushed optic nerve and into the optic chiasm. It also had neuroprotective effects on axotomized RGCs. Interestingly, the strong stimulation of neurite

Acknowledgments

This work was supported by the Swiss National Science Foundation (SNF) grant nr. 31-122527/1 and the SNF National Center of Competence in Research ‘Neural Plasticity and Repair’. We thank Dr Olivier Raineteau for sharing his Leica SPE-II confocal microscope with us. The authors declare no competing financial interests.

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