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-A^flox/flox) and neuron-specific (Thy1-Cre^tg+xRtn4^flox/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 Rtn4^flox/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.In the adult mammalian central nervous system (CNS), axons have a very limited capacity to regenerate after traumatic injury. This lack of axonal regeneration is thought to be mainly due to the presence of growth-inhibiting molecules in the injured CNS environment^{1, 2} and due to the low intrinsic growth capacity of mature neurons.³Nogo-A is a well-studied inhibitory protein for axonal growth, plasticity and regeneration after CNS injury.^{4, 5} Nogo-A is predominantly expressed in oligodendrocytes in the adult CNS, where it is thought to stabilize the neuronal circuits in healthy conditions and to inhibit neurite growth and plasticity after lesion.² Neutralizing Nogo-A by function-blocking antibodies or genetic knockout (KO) has been shown to improve axonal sprouting and regeneration in the injured spinal cord and brain.^{6, 7, 8, 9, 10, 11}In addition to oligodendrocytes and myelin, Nogo-A is expressed in growing and immature neurons, as well as in some adult neurons.^{12, 13} Neurons express Nogo-A receptors such as the Nogo-66 receptor 1 (NgR1)¹⁴ and the Nogo-A-Δ20-specific sphingosine 1-phosphate receptor 2 (S1PR2).¹⁵ They can co-express them along with Nogo-A,¹³ an observation that raises the possibility of cis-interactions between the ligand and its receptors within or at the cell surface of the same cell. This mechanism has previously been described for axonal guidance molecules such as Ephrins and Semaphorins, and could have a major role in the neuronal response to extracellular growth inhibitors during development.^{16, 17}In the adult CNS, the expression of neuronal Nogo-A remains elevated mainly in plastic regions such as in the hippocampus, olfactory bulb or neocortex, and in the dorsal root ganglia.¹² Nogo-A and NgR1 were shown to regulate synaptic plasticity, for example, long-term potentiation in the hippocampus and in the sensory-motor cortex,^{18, 19, 20, 21, 22} whereas the effects of neuronal Nogo-A after injury are not yet well understood. During development, neuronal Nogo-A influences neuronal migration,^{23, 24} survival,^{25, 26} cell spreading and neurite growth.^{27, 28} In injured adult retinal ganglion cells (RGCs), silencing neuronal Nogo-A resulted in a marked reduction of regenerative sprouting and decreased expression of growth-associated molecules.²⁹ Furthermore, in the optic nerve, axonal regeneration was not improved in conventional Nogo-A KO animals, in which both glial and neuronal Nogo-A were deleted.²⁹ The present study therefore aimed to investigate whether glial and neuronal Nogo-A differently influence axonal growth in vivo using cell type-specific Nogo-A KO mouse lines and adeno-associated virus (AAV)-mediated recombination of the Nogo-A gene in neurons. The results show that significantly more axons grew through the lesion site in the oligodendrocyte-specific Nogo-A KO mice. In contrast, neuron-specific ablation of Nogo-A in RGCs reduced the number of regenerating axons after optic nerve crush injury (ONC). In summary, we show that inactivating Nogo-A specifically in oligodendrocytes appears to be the most successful strategy to promote axonal regeneration in the adult optic nerve.