Substrate properties of zebrafish Rtn4b/Nogo and axon regeneration in the zebrafish optic nerve

Abstract

This study explored why lesioned retinal ganglion cell (RGC) axons regenerate successfully in the zebrafish optic nerve despite the presence of Rtn4b, the homologue of the rat neurite growth inhibitor RTN4-A/Nogo-A. Rat Nogo-A and zebrafish Rtn4b possess characteristic motifs (M1-4) in the Nogo-A-specific region, which contains delta20, the most inhibitory region of rat Nogo-A. To determine whether zebrafish M1-4 is inhibitory as rat M1-4 and Nogo-A delta20, proteins were recombinantly expressed and used as substrates for zebrafish single cell RGCs, mouse hippocampal neurons and goldfish, zebrafish and chick retinal explants. When offered as homogenous substrates, neurites of hippocampal neurons and of zebrafish single cell RGCs were inhibited by zebrafish M1-4, rat M1-4, and Nogo-A delta20. Neurite length increased when zebrafish single cell RGCs were treated with receptor-type-specific antagonists and, respectively, with morpholinos (MO) against S1PR2 and S1PR5a-which represent candidate zebrafish Nogo-A receptors. In a stripe assay, however, where M1-4 lanes alternate with polylysine-(Plys)-only lanes, RGC axons from goldfish, zebrafish, and chick retinal explants avoided rat M1-4 but freely crossed zebrafish M1-4 lanes-suggesting that zebrafish M1-4 is growth permissive and less inhibitory than rat M1-4. Moreover, immunostainings and dot blots of optic nerve and myelin showed that expression of Rtn4b is very low in tissue and myelin at 3-5 days after lesion when axons regenerate. Thus, Rtn4b seems to represent no major obstacle for axon regeneration in vivo because it is less inhibitory for RGC axons from retina explants, and because of its low abundance.

Application of CNTF or FGF-2 increases the number of M2-like macrophages after optic nerve injury in adult Rana pipiens

“…However, Xenopus optic tract myelin is not inhibitory to axonal extension (unlike that from the spinal cord)[61], despite both expressing an ortholog of Nogo [62]. It is more likely that, as in fish [5,63,64], the relatively few frog oligodendrocytes and their myelin are not inhibitory.…”

Dynamic cell interactions allow spinal cord regeneration in zebrafish

“…In zebrafish, the Nogo homolog rtn4b has a similar domain structure to its mammalian counterpart, but the most inhibitory domain of the protein (delta20) is less inhibitory in cross-species in vitro assays for the zebrafish protein compared to the mammalian version of the protein [28]. At the same time, zebrafish oligodendrocytes produce axon-growth promoting cell surface molecules, such as mpz [29] and l1cam [30], at increased levels after injury.…”

Application of Antibodies to Neuronally Expressed Nogo-A Increases Neuronal Survival and Neurite Outgrowth

“…Our results with rodent Nogo-A are in agreement with observations on regeneration-competent zebrafish which upregulate Nogo-A homolog expression in retinal ganglion cells after an optic nerve lesion, an effect that could be inhibited in vivo by antisense oligonucleotides targeting Nogo-A [ 32 ]. In addition to the intrinsic capability of retinal ganglion cells to regrow severed axons, zebrafish oligodendrocytes, and myelin-expressing Nogo-A lack the inhibitory functions of rodent myelin when offered, for instance, as a substrate barrier to neurites [ 32 , 33 ]. We conclude that intrinsic neuronal expression of Nogo-A is an important ingredient in regeneration after injury and expect that small compounds triggering the beneficial functions of neuronally-expressed Nogo-A will contribute to the amelioration of the severe consequences of central nervous system injury.…”

“…Purkinje cells are destroyed during cell preparation and do not survive, even after only 2 to 3 days of cell culture. Cerebellar granule neurons were prepared from 6- to 8-day-old mice as described by us [ 33 ]. In brief, the cerebellar cortices were cut into small pieces, which were then dissociated with trypsin and DNase for 15 min, washed with Hank’s balanced salt solution (HBSS), and then centrifuged at 100× g for 15 min at 4 °C.…”