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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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Rtn4b MO-induced reduction in axon regeneration in thein vivoregeneration assays. (A-F) After application of Alexa488 to the regenerating axons (distal from the original lesion and MO application site), the retrogradely labeled RGCs are counted in retina whole mounts. Many more Alexa488-labeled RGCs are recognized 9 days after ONS and control (Co) MO application (A) than on the contralateral retina (D) belonging to the nerve that received Rtn4b MO1 (or MO2). (B, E) The RGCs contain lissamine associated with the MOs. (C,F) Merge of (A,B) and (D,E). Scale bar, 50 μm. (G) The histogram demonstrates the decline in the number of Alexa-labeled RGCs 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. Three different experiments with n, 10 retinal squares (300 × 300 μm) for each experimental group were statistically evaluated using Student’s T-test. The differences between groups are statistically significantly different, ***P < 0.001.
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Fig6: Rtn4b MO-induced reduction in axon regeneration in thein vivoregeneration assays. (A-F) After application of Alexa488 to the regenerating axons (distal from the original lesion and MO application site), the retrogradely labeled RGCs are counted in retina whole mounts. Many more Alexa488-labeled RGCs are recognized 9 days after ONS and control (Co) MO application (A) than on the contralateral retina (D) belonging to the nerve that received Rtn4b MO1 (or MO2). (B, E) The RGCs contain lissamine associated with the MOs. (C,F) Merge of (A,B) and (D,E). Scale bar, 50 μm. (G) The histogram demonstrates the decline in the number of Alexa-labeled RGCs 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. Three different experiments with n, 10 retinal squares (300 × 300 μm) for each experimental group were statistically evaluated using Student’s T-test. The differences between groups are statistically significantly different, ***P < 0.001.

Mentions: In a second assay, the optic nerve of fish after ONS and MO treatment was re-sectioned at 7 days, 2 to 3 mm distal from the first lesion, and Alexa488-dextran was applied to retrogradely label RGCs with regenerating axons [17]. Two days later, the dextran-labeled RGCs were counted in left and right retina whole mounts (left side: Rtn4b MO1 and Rtn4b MO2, respectively; right side: control MO) in seven independent experiments (Figure 6A, B, C, D, E, F). The number of dextran-labeled RGCs was on average reduced by 54% over controls with MO1 (P < 0.001) and by 62% with MO2 (P < 0.001) (Figure 6G). Thus, downregulation of Rtn4b significantly blocks RGC axon regeneration.Figure 6


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)

Rtn4b MO-induced reduction in axon regeneration in thein vivoregeneration assays. (A-F) After application of Alexa488 to the regenerating axons (distal from the original lesion and MO application site), the retrogradely labeled RGCs are counted in retina whole mounts. Many more Alexa488-labeled RGCs are recognized 9 days after ONS and control (Co) MO application (A) than on the contralateral retina (D) belonging to the nerve that received Rtn4b MO1 (or MO2). (B, E) The RGCs contain lissamine associated with the MOs. (C,F) Merge of (A,B) and (D,E). Scale bar, 50 μm. (G) The histogram demonstrates the decline in the number of Alexa-labeled RGCs 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. Three different experiments with n, 10 retinal squares (300 × 300 μm) for each experimental group were statistically evaluated using Student’s T-test. The differences between groups are statistically significantly different, ***P < 0.001.
© Copyright Policy - open-access
Related In: Results  -  Collection

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Fig6: Rtn4b MO-induced reduction in axon regeneration in thein vivoregeneration assays. (A-F) After application of Alexa488 to the regenerating axons (distal from the original lesion and MO application site), the retrogradely labeled RGCs are counted in retina whole mounts. Many more Alexa488-labeled RGCs are recognized 9 days after ONS and control (Co) MO application (A) than on the contralateral retina (D) belonging to the nerve that received Rtn4b MO1 (or MO2). (B, E) The RGCs contain lissamine associated with the MOs. (C,F) Merge of (A,B) and (D,E). Scale bar, 50 μm. (G) The histogram demonstrates the decline in the number of Alexa-labeled RGCs 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. Three different experiments with n, 10 retinal squares (300 × 300 μm) for each experimental group were statistically evaluated using Student’s T-test. The differences between groups are statistically significantly different, ***P < 0.001.
Mentions: In a second assay, the optic nerve of fish after ONS and MO treatment was re-sectioned at 7 days, 2 to 3 mm distal from the first lesion, and Alexa488-dextran was applied to retrogradely label RGCs with regenerating axons [17]. Two days later, the dextran-labeled RGCs were counted in left and right retina whole mounts (left side: Rtn4b MO1 and Rtn4b MO2, respectively; right side: control MO) in seven independent experiments (Figure 6A, B, C, D, E, F). The number of dextran-labeled RGCs was on average reduced by 54% over controls with MO1 (P < 0.001) and by 62% with MO2 (P < 0.001) (Figure 6G). Thus, downregulation of Rtn4b significantly blocks RGC axon regeneration.Figure 6

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