Franziska Christ

Systemic deletion of the myelin-associated outgrowth inhibitor Nogo-A improves regenerative and plastic responses after spinal cord injury.

To investigate the role of the myelin-associated protein Nogo-A on axon sprouting and regeneration in the adult central nervous system (CNS), we generated Nogo-A-deficient mice. Nogo-A knockout (KO) mice were viable, fertile, and not obviously afflicted by major developmental or neurological disturbances. The shorter splice form Nogo-B was strongly upregulated in the CNS. The inhibitory effect of spinal cord extract for growing neurites was decreased in the KO mice. Two weeks following adult dorsal hemisection of the thoracic spinal cord, Nogo-A KO mice displayed more corticospinal tract (CST) fibers growing toward and into the lesion compared to their wild-type littermates. CST fibers caudal to the lesion-regenerating and/or sprouting from spared intact fibers-were also found to be more frequent in Nogo-A-deficient animals.

Nogo-A and Myelin-Associated Glycoprotein Differently Regulate Oligodendrocyte Maturation and Myelin Formation

Nogo-A is one of the most potent oligodendrocyte-derived inhibitors for axonal regrowth in the injured adult CNS. However, the physiological function of Nogo-A in development and in healthy oligodendrocytes is still unknown. In the present study, we investigated the role of Nogo-A for myelin formation in the developing optic nerve. By quantitative real-time PCR, we found that the expression of Nogo-A increased faster in differentiating oligodendrocytes than that of the major myelin proteins MBP (myelin basic protein), PLP (proteolipid protein)/DM20, and CNP (2′,3′-cyclic nucleotide 3′-phosphodiesterase). The analysis of optic nerves and cerebella of mice deficient for Nogo-A (Nogo-A−/−) revealed a marked delay of oligodendrocyte differentiation, myelin sheath formation, and axonal caliber growth within the first postnatal month. The combined deletion of Nogo-A and MAG caused a more severe transient hypomyelination. In contrast to MAG−/− mice, Nogo-A−/− mutants did not present abnormalities in the structure of myelin sheaths and Ranvier nodes. The common binding protein for Nogo-A and MAG, NgR1, was exclusively upregulated in MAG−/− animals, whereas the level of Lingo-1, a coreceptor, remained unchanged. Together, our results demonstrate that Nogo-A and MAG are differently involved in oligodendrocyte maturation in vivo, and suggest that Nogo-A may influence also remyelination in pathological conditions such as multiple sclerosis.

Nogo-A expressed in Schwann cells impairs axonal regeneration after peripheral nerve injury

Înjured axons in mammalian peripheral nerves often regenerate successfully over long distances, in contrast to axons in the brain and spinal cord (CNS). Neurite growth-inhibitory proteins, including the recently cloned membrane protein Nogo-A, are enriched in the CNS, in particular in myelin. Nogo-A is not detectable in peripheral nerve myelin. Using regulated transgenic expression of Nogo-A in peripheral nerve Schwann cells, we show that axonal regeneration and functional recovery are impaired after a sciatic nerve crush. Nogo-A thus overrides the growth-permissive and -promoting effects of the lesioned peripheral nerve, demonstrating its in vivo potency as an inhibitor of axonal regeneration.

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

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.

Neuronal Nogo-A upregulation does not contribute to ER stress-associated apoptosis but participates in the regenerative response in the axotomized adult retina

Nogo-A, an axonal growth inhibitory protein known to be mostly present in CNS myelin, was upregulated in retinal ganglion cells (RGCs) after optic nerve injury in adult mice. Nogo-A increased concomitantly with the endoplasmic reticulum stress (ER stress) marker C/EBP homologous protein (CHOP), but CHOP immunostaining and the apoptosis marker annexin V did not co-localize with Nogo-A in individual RGC cell bodies, suggesting that injury-induced Nogo-A upregulation is not involved in axotomy-induced cell death. Silencing Nogo-A with an adeno-associated virus serotype 2 containing a short hairpin RNA (AAV2.shRNA-Nogo-A) or Nogo-A gene ablation in knock-out (KO) animals had little effect on the lesion-induced cell stress or death. On the other hand, Nogo-A overexpression mediated by AAV2.Nogo-A exacerbated RGC cell death after injury. Strikingly, however, injury-induced sprouting of the cut axons and the expression of growth-associated molecules were markedly reduced by AAV2.shRNA-Nogo-A. The axonal growth in the optic nerve activated by the intraocular injection of the inflammatory molecule Pam3Cys tended to be lower in Nogo-A KO mice than in WT mice. Nogo-A overexpression in RGCs in vivo or in the neuronal cell line F11 in vitro promoted regeneration, demonstrating a positive, cell-autonomous role for neuronal Nogo-A in the modulation of axonal regeneration.

Cell type-specific Nogo-A gene ablation promotes axonal regeneration in the injured adult optic nerve.

Nogo-A is a well-known myelin-enriched inhibitory protein for axonal growth and regeneration in the central nervous system (CNS). Besides oligodendrocytes, our previous data revealed that Nogo-A is also expressed in subpopulations of neurons including retinal ganglion cells, in which it can have a positive role in the neuronal growth response after injury, through an unclear mechanism. In the present study, we analyzed the opposite roles of glial versus neuronal Nogo-A in the injured visual system. To this aim, we created oligodendrocyte (Cnp-Cre+/−xRtn4/Nogo-Aflox/flox) and neuron-specific (Thy1-Cretg+xRtn4flox/flox) conditional Nogo-A knock-out (KO) mouse lines. Following complete intraorbital optic nerve crush, both spontaneous and inflammation-mediated axonal outgrowth was increased in the optic nerves of the glia-specific Nogo-A KO mice. In contrast, neuron-specific deletion of Nogo-A in a KO mouse line or after acute gene recombination in retinal ganglion cells mediated by adeno-associated virus serotype 2.Cre virus injection in Rtn4flox/flox animals decreased axon sprouting in the injured optic nerve. These results therefore show that selective ablation of Nogo-A in oligodendrocytes and myelin in the optic nerve is more effective at enhancing regrowth of injured axons than what has previously been observed in conventional, complete Nogo-A KO mice. Our data also suggest that neuronal Nogo-A in retinal ganglion cells could participate in enhancing axonal sprouting, possibly by cis-interaction with Nogo receptors at the cell membrane that may counteract trans-Nogo-A signaling. We propose that inactivating Nogo-A in glia while preserving neuronal Nogo-A expression may be a successful strategy to promote axonal regeneration in the CNS.

The growth-associated protein GAP-43 is specifically expressed in tyrosine hydroxylase-positive cells of the rat retina

In the adult retina, the growth-associated protein GAP-43 is exclusively present in three distinct sublaminae of the inner plexiform layer. During postnatal development, it is transiently expressed in the optic nerve fibers. No conclusions about the GAP-43 expressing cells can be derived from immunohistochemical stainings because GAP-43 protein is rapidly transported into the distal neuronal processes. We have combined immunohistochemistry to study the protein expression of GAP-43 and non-radioactive in situ hybridization to study the cellular expression of GAP-43 in the rat retina. We have found that in the mature retina GAP-43 mRNA is present only in retinal ganglion cells and in a small subset of cells of the inner nuclear layer. During postnatal development, no cells besides retinal ganglion cells and a subpopulation of cells in the inner nuclear layer express GAP-43 mRNA. Double staining experiments with tyrosine hydroxylase (TH) immunohistochemistry and GAP-43 in situ hybridization showed that GAP-43 expressing cells in the inner nuclear layer are immunoreactive for TH. They are most probably dopaminergic amacrine cells. Our results show that GAP-43 expression in the retina is restricted to very few cell types. They suggest that TH-positive cells (probably dopaminergic amacrine cells) retain a higher degree of structural plasticity in the adult retina.