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Upregulation of the zebrafish Nogo-A homologue, Rtn4b, in retinal ganglion cells is functionally involved in axon regeneration.

Welte C, Engel S, Stuermer CA - Neural Dev (2015)

Bottom Line: MO1 and MO2 reduced the number of axons from retina explants in a concentration-dependent manner.With MO1, the reduction was 55% (70 μM MO1) and 74% (140 μM MO1), respectively, with MO2: 59% (70 μM MO2) and 73% (140 μM MO2), respectively (compared to the control MO-treated side).The spontaneous lesion-induced upregulation of Rtn4b in fish correlates with an increase in ER, soma size, biosynthetic activity, and thus growth and predicts that mammalian neurons require the same upregulation in order to successfully regenerate RGC axons.

View Article: PubMed Central - PubMed

Affiliation: Department of Biology, University of Konstanz, Universitätsstraße 10, 78457, Konstanz, Germany. Cornelia.welte@uni-konstanz.de.

ABSTRACT

Background: In contrast to mammals, zebrafish successfully regenerate retinal ganglion cell (RGC) axons after optic nerve section (ONS). This difference is explained on the one hand by neurite growth inhibitors in mammals (including Nogo-A), as opposed to growth-promoting glial cells in the fish visual pathway, and on the other hand by the neuron-intrinsic properties allowing the upregulation of growth-associated proteins in fish RGCs but not in mammals.

Results: Here, we report that Rtn4b, the zebrafish homologue of mammalian Nogo-A/RTN4-A, is upregulated in axotomized zebrafish RGCs and is primarily associated with the endoplasmic reticulum (ER). Rtn4b functions as a neuron-intrinsic determinant for axon regeneration, as was shown by downregulating Rtn4b through retrogradely transported morpholinos (MOs), applied to the optic nerve at the time of ONS. MO1 and MO2 reduced the number of axons from retina explants in a concentration-dependent manner. With MO1, the reduction was 55% (70 μM MO1) and 74% (140 μM MO1), respectively, with MO2: 59% (70 μM MO2) and 73% (140 μM MO2), respectively (compared to the control MO-treated side). Moreover, regenerating axons 7d after ONS and MO1 or MO2 application were labeled by Alexa488, applied distal to the first lesion. The number of Alexa488 labeled RGCs, containing the Rtn4b MO1 or MO2, was reduced by 54% and 62%, respectively, over control MO.

Conclusions: Thus, Rtn4b is an important neuron-intrinsic component and required for the success of axon regeneration in the zebrafish visual system. The spontaneous lesion-induced upregulation of Rtn4b in fish correlates with an increase in ER, soma size, biosynthetic activity, and thus growth and predicts that mammalian neurons require the same upregulation in order to successfully regenerate RGC axons.

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Rtn4a and Rtn4b staining of ER in zebrafish oligodendrocytes. (A) Labeling of fixed zebrafish oligodendrocytes with Rtn4b reveals a reticular structure similar to the ER staining with CLIMP63 AB (B). (C) Rtn4a AB also labels ER in oligodendrocytes. (D) Exposing live oligodendrocytes to Rtn4b AB gives no cell surface staining. DAPI stains the nuclei in (A,D) and also in (B,C) where, however, the red ER stain covers the blue. (E) Zebrafish RGC axons and growth cones are labeled by the Rtn4b AB. These structures are also labeled with CLIMP63 AB (F). Scale bar, 10 μm.
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Fig2: Rtn4a and Rtn4b staining of ER in zebrafish oligodendrocytes. (A) Labeling of fixed zebrafish oligodendrocytes with Rtn4b reveals a reticular structure similar to the ER staining with CLIMP63 AB (B). (C) Rtn4a AB also labels ER in oligodendrocytes. (D) Exposing live oligodendrocytes to Rtn4b AB gives no cell surface staining. DAPI stains the nuclei in (A,D) and also in (B,C) where, however, the red ER stain covers the blue. (E) Zebrafish RGC axons and growth cones are labeled by the Rtn4b AB. These structures are also labeled with CLIMP63 AB (F). Scale bar, 10 μm.

Mentions: The affinity purified antiserum against zebrafish Rtn4b [16] labeled all retinal layers but was brighter over RGC somata compared to other retinal neurons (Figure 1A). The RGC axon layer which was intensely labeled by the anti-MBP antibody (AB) (fish RGC axons are myelinated in their intraretinal path) was only weakly stained by the Rtn4b AB (Figure 1A, B, C). Ten days after ONS, RGC somata had significantly increased expression of Rtn4b indicating that ONS leads to Rtn4b upregulation in neurons (Figure 1B). In the normal optic nerve, Rtn4b labeling was weak (Figure 1D) whereas anti-MBP AB strongly labeled the myelin (Figure 1F, M) in the normal nerve and after ONS. The staining with Rtn4a AB was similar to MBP, but the AB labeled in addition the boundaries of axon fascicles and further subdivisions of the fascicles (Figure 1E). Rtn4a therefore appears to reside in astrocytic structures as suggested earlier [18] and myelin. In the nerve 10 days after ONS, Rtn4b labeling was associated with glial cell processes around fascicles and more strikingly with regenerating RGC axons which were identified by anti-neurolin AB [19] (Figure 1G, H, I, P). Accordingly, axons and growth cones in culture were also labeled (Figure 2E). Rtn4a AB also stains RGC growth cones in vitro [18] but in sections through the nerve strongly stained the fascicle boundaries and subdivisions rather than neurolin-positive regenerating axons (Figure 1J, K, L, Q). In the nerve 10 days after ONS, myelin detected by MBP AB was intense and the neurolin-positive regenerating axons were located amidst the myelin staining (Figure 1M, N, O, R). Together, this staining shows that regenerating RGC axons in the nerve and in vitro are Rtn4b-positive and cross through MBP-labeled myelin. Rtn4a is in myelin and astrocytic fascicle boundaries and subdivisions but not to the same extent in neurolin-positive axons as Rtn4b. Rtn4b appears less prominent in CNS myelin in the retina and optic nerve but is significantly upregulated in RGCs and RGC axons after ONS.Figure 1


Upregulation of the zebrafish Nogo-A homologue, Rtn4b, in retinal ganglion cells is functionally involved in axon regeneration.

Welte C, Engel S, Stuermer CA - Neural Dev (2015)

Rtn4a and Rtn4b staining of ER in zebrafish oligodendrocytes. (A) Labeling of fixed zebrafish oligodendrocytes with Rtn4b reveals a reticular structure similar to the ER staining with CLIMP63 AB (B). (C) Rtn4a AB also labels ER in oligodendrocytes. (D) Exposing live oligodendrocytes to Rtn4b AB gives no cell surface staining. DAPI stains the nuclei in (A,D) and also in (B,C) where, however, the red ER stain covers the blue. (E) Zebrafish RGC axons and growth cones are labeled by the Rtn4b AB. These structures are also labeled with CLIMP63 AB (F). Scale bar, 10 μm.
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Fig2: Rtn4a and Rtn4b staining of ER in zebrafish oligodendrocytes. (A) Labeling of fixed zebrafish oligodendrocytes with Rtn4b reveals a reticular structure similar to the ER staining with CLIMP63 AB (B). (C) Rtn4a AB also labels ER in oligodendrocytes. (D) Exposing live oligodendrocytes to Rtn4b AB gives no cell surface staining. DAPI stains the nuclei in (A,D) and also in (B,C) where, however, the red ER stain covers the blue. (E) Zebrafish RGC axons and growth cones are labeled by the Rtn4b AB. These structures are also labeled with CLIMP63 AB (F). Scale bar, 10 μm.
Mentions: The affinity purified antiserum against zebrafish Rtn4b [16] labeled all retinal layers but was brighter over RGC somata compared to other retinal neurons (Figure 1A). The RGC axon layer which was intensely labeled by the anti-MBP antibody (AB) (fish RGC axons are myelinated in their intraretinal path) was only weakly stained by the Rtn4b AB (Figure 1A, B, C). Ten days after ONS, RGC somata had significantly increased expression of Rtn4b indicating that ONS leads to Rtn4b upregulation in neurons (Figure 1B). In the normal optic nerve, Rtn4b labeling was weak (Figure 1D) whereas anti-MBP AB strongly labeled the myelin (Figure 1F, M) in the normal nerve and after ONS. The staining with Rtn4a AB was similar to MBP, but the AB labeled in addition the boundaries of axon fascicles and further subdivisions of the fascicles (Figure 1E). Rtn4a therefore appears to reside in astrocytic structures as suggested earlier [18] and myelin. In the nerve 10 days after ONS, Rtn4b labeling was associated with glial cell processes around fascicles and more strikingly with regenerating RGC axons which were identified by anti-neurolin AB [19] (Figure 1G, H, I, P). Accordingly, axons and growth cones in culture were also labeled (Figure 2E). Rtn4a AB also stains RGC growth cones in vitro [18] but in sections through the nerve strongly stained the fascicle boundaries and subdivisions rather than neurolin-positive regenerating axons (Figure 1J, K, L, Q). In the nerve 10 days after ONS, myelin detected by MBP AB was intense and the neurolin-positive regenerating axons were located amidst the myelin staining (Figure 1M, N, O, R). Together, this staining shows that regenerating RGC axons in the nerve and in vitro are Rtn4b-positive and cross through MBP-labeled myelin. Rtn4a is in myelin and astrocytic fascicle boundaries and subdivisions but not to the same extent in neurolin-positive axons as Rtn4b. Rtn4b appears less prominent in CNS myelin in the retina and optic nerve but is significantly upregulated in RGCs and RGC axons after ONS.Figure 1

Bottom Line: MO1 and MO2 reduced the number of axons from retina explants in a concentration-dependent manner.With MO1, the reduction was 55% (70 μM MO1) and 74% (140 μM MO1), respectively, with MO2: 59% (70 μM MO2) and 73% (140 μM MO2), respectively (compared to the control MO-treated side).The spontaneous lesion-induced upregulation of Rtn4b in fish correlates with an increase in ER, soma size, biosynthetic activity, and thus growth and predicts that mammalian neurons require the same upregulation in order to successfully regenerate RGC axons.

View Article: PubMed Central - PubMed

Affiliation: Department of Biology, University of Konstanz, Universitätsstraße 10, 78457, Konstanz, Germany. Cornelia.welte@uni-konstanz.de.

ABSTRACT

Background: In contrast to mammals, zebrafish successfully regenerate retinal ganglion cell (RGC) axons after optic nerve section (ONS). This difference is explained on the one hand by neurite growth inhibitors in mammals (including Nogo-A), as opposed to growth-promoting glial cells in the fish visual pathway, and on the other hand by the neuron-intrinsic properties allowing the upregulation of growth-associated proteins in fish RGCs but not in mammals.

Results: Here, we report that Rtn4b, the zebrafish homologue of mammalian Nogo-A/RTN4-A, is upregulated in axotomized zebrafish RGCs and is primarily associated with the endoplasmic reticulum (ER). Rtn4b functions as a neuron-intrinsic determinant for axon regeneration, as was shown by downregulating Rtn4b through retrogradely transported morpholinos (MOs), applied to the optic nerve at the time of ONS. MO1 and MO2 reduced the number of axons from retina explants in a concentration-dependent manner. With MO1, the reduction was 55% (70 μM MO1) and 74% (140 μM MO1), respectively, with MO2: 59% (70 μM MO2) and 73% (140 μM MO2), respectively (compared to the control MO-treated side). Moreover, regenerating axons 7d after ONS and MO1 or MO2 application were labeled by Alexa488, applied distal to the first lesion. The number of Alexa488 labeled RGCs, containing the Rtn4b MO1 or MO2, was reduced by 54% and 62%, respectively, over control MO.

Conclusions: Thus, Rtn4b is an important neuron-intrinsic component and required for the success of axon regeneration in the zebrafish visual system. The spontaneous lesion-induced upregulation of Rtn4b in fish correlates with an increase in ER, soma size, biosynthetic activity, and thus growth and predicts that mammalian neurons require the same upregulation in order to successfully regenerate RGC axons.

Show MeSH
Related in: MedlinePlus