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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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Quantification of axon number after MO application in the outgrowth assay. (A,B) Retina mini-explants isolated from retinae 5 days after (A) control MO (Co) or (B) Rtn4b MO1 application to the optic nerve, extend axons and axon fascicles (arrows) after 24 h in vitro. Outgrowth is significantly reduced on the Rtn4b MO-treated side. Scale bar, 100 μm. (C) The histogram demonstrates the decline in the number of axons extending from retina explants in vitro after MO1 and MO2 application to the optic nerve, in comparison to axon number from control (Co) MO-treated fish (100%). Bars indicate standard deviation. The differences between groups are statistically significantly different. Quantification was done on three replicates from three different experiments, and for statistical analysis, Student’s T-test was used. *P < 0.05, **P < 0.01.
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Fig5: Quantification of axon number after MO application in the outgrowth assay. (A,B) Retina mini-explants isolated from retinae 5 days after (A) control MO (Co) or (B) Rtn4b MO1 application to the optic nerve, extend axons and axon fascicles (arrows) after 24 h in vitro. Outgrowth is significantly reduced on the Rtn4b MO-treated side. Scale bar, 100 μm. (C) The histogram demonstrates the decline in the number of axons extending from retina explants in vitro after MO1 and MO2 application to the optic nerve, in comparison to axon number from control (Co) MO-treated fish (100%). Bars indicate standard deviation. The differences between groups are statistically significantly different. Quantification was done on three replicates from three different experiments, and for statistical analysis, Student’s T-test was used. *P < 0.05, **P < 0.01.

Mentions: Next, we analyzed whether Rtn4b MOs impair axon regeneration. In the so-called outgrowth assay, the retinae were divided into mini-explants at 5 days after ONS and MO application and seeded onto pLys-coated coverslips. Counts of axons in Rtn4b MO-treated compared to control MO-treated retinae at 24 h shows that Rtn4b downregulation reduced axon outgrowth significantly. MO1-treated retinae at 70 and 140 μM, respectively, gave a reduction in number of axons of 55% and 74% over controls (Figure 5). MO2-treated retinae at 70 and 140 μM, respectively, showed a 59% and 73% reduction over controls (80 explants were evaluated per experiment). This reduction in axon number was statistically significant with MO1 at 70 μM (P < 0.05), with MO2 at 70 μM (P < 0.01) and at 140 μM MO1 (P < 0.05) and MO2 (P < 0.01). Thus, downregulation of Rtn4b with two unrelated MOs blocks RGC axon outgrowth in a concentration-dependent manner.Figure 5


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)

Quantification of axon number after MO application in the outgrowth assay. (A,B) Retina mini-explants isolated from retinae 5 days after (A) control MO (Co) or (B) Rtn4b MO1 application to the optic nerve, extend axons and axon fascicles (arrows) after 24 h in vitro. Outgrowth is significantly reduced on the Rtn4b MO-treated side. Scale bar, 100 μm. (C) The histogram demonstrates the decline in the number of axons extending from retina explants in vitro after MO1 and MO2 application to the optic nerve, in comparison to axon number from control (Co) MO-treated fish (100%). Bars indicate standard deviation. The differences between groups are statistically significantly different. Quantification was done on three replicates from three different experiments, and for statistical analysis, Student’s T-test was used. *P < 0.05, **P < 0.01.
© Copyright Policy - open-access
Related In: Results  -  Collection

License 1 - License 2
Show All Figures
getmorefigures.php?uid=PMC4374419&req=5

Fig5: Quantification of axon number after MO application in the outgrowth assay. (A,B) Retina mini-explants isolated from retinae 5 days after (A) control MO (Co) or (B) Rtn4b MO1 application to the optic nerve, extend axons and axon fascicles (arrows) after 24 h in vitro. Outgrowth is significantly reduced on the Rtn4b MO-treated side. Scale bar, 100 μm. (C) The histogram demonstrates the decline in the number of axons extending from retina explants in vitro after MO1 and MO2 application to the optic nerve, in comparison to axon number from control (Co) MO-treated fish (100%). Bars indicate standard deviation. The differences between groups are statistically significantly different. Quantification was done on three replicates from three different experiments, and for statistical analysis, Student’s T-test was used. *P < 0.05, **P < 0.01.
Mentions: Next, we analyzed whether Rtn4b MOs impair axon regeneration. In the so-called outgrowth assay, the retinae were divided into mini-explants at 5 days after ONS and MO application and seeded onto pLys-coated coverslips. Counts of axons in Rtn4b MO-treated compared to control MO-treated retinae at 24 h shows that Rtn4b downregulation reduced axon outgrowth significantly. MO1-treated retinae at 70 and 140 μM, respectively, gave a reduction in number of axons of 55% and 74% over controls (Figure 5). MO2-treated retinae at 70 and 140 μM, respectively, showed a 59% and 73% reduction over controls (80 explants were evaluated per experiment). This reduction in axon number was statistically significant with MO1 at 70 μM (P < 0.05), with MO2 at 70 μM (P < 0.01) and at 140 μM MO1 (P < 0.05) and MO2 (P < 0.01). Thus, downregulation of Rtn4b with two unrelated MOs blocks RGC axon outgrowth in a concentration-dependent manner.Figure 5

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