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Active mechanistic target of rapamycin plays an ancillary rather than essential role in zebrafish CNS axon regeneration.

Diekmann H, Kalbhen P, Fischer D - Front Cell Neurosci (2015)

Bottom Line: Remarkably, regulation of mTOR activity after optic nerve injury in zebrafish is fundamentally different compared to mammals.Moreover, inhibition of mTOR using rapamycin significantly reduced axon regeneration in vivo and compromised functional recovery after optic nerve injury.Therefore, axotomy-induced mTOR activity is involved in CNS axon regeneration in zebrafish similar to mammals, although it plays an ancillary rather than essential role in this regeneration-competent species.

View Article: PubMed Central - PubMed

Affiliation: Division of Experimental Neurology, Department of Neurology, Heinrich-Heine-University of Düsseldorf Düsseldorf, Germany.

ABSTRACT
The developmental decrease of the intrinsic regenerative ability of the mammalian central nervous system (CNS) is associated with reduced activity of mechanistic target of rapamycin (mTOR) in mature neurons such as retinal ganglion cells (RGCs). While mTOR activity is further decreased upon axonal injury, maintenance of its pre-injury level, for instance by genetic deletion of the phosphatase and tensin homolog (PTEN), markedly promotes axon regeneration in mammals. The current study now addressed the question whether active mTOR might generally play a central role in axon regeneration by analyzing its requirement in regeneration-competent zebrafish. Remarkably, regulation of mTOR activity after optic nerve injury in zebrafish is fundamentally different compared to mammals. Hardly any activity was detected in naïve RGCs, whereas it was markedly increased upon axotomy in vivo as well as in dissociated cell cultures. After a short burst, mTOR activity was quickly attenuated, which is contrary to the requirements for axon regeneration in mammals. Surprisingly, mTOR activity was not essential for axonal growth per se, but correlated with cytokine- and PTEN inhibitor-induced neurite extension in vitro. Moreover, inhibition of mTOR using rapamycin significantly reduced axon regeneration in vivo and compromised functional recovery after optic nerve injury. Therefore, axotomy-induced mTOR activity is involved in CNS axon regeneration in zebrafish similar to mammals, although it plays an ancillary rather than essential role in this regeneration-competent species.

No MeSH data available.


Related in: MedlinePlus

Compromised axon regeneration and functional recovery upon mTOR inhibition. (A,B) Maximum intensity projections (85 × 0.9 μm confocal z-sections) of wholemount optic nerves from DMSO- (A) and 0.2 μM rapamycin (Rap, B) -treated GAP43::GFP zebrafish at 2.5 days post injury (dpi). The lesion site is indicated with a dashed line, proximal is to the left. Scale bar = 200 μm. (A′,B′) Higher magnifications of the boxed areas in (A) and (B), respectively, using maximum intensity projections of 10 × 0.9 μm confocal stacks. Scale bar = 50 μm. (C) Quantification of axon profiles per mm optic nerve diameter on single z-sections at 200 and 500 μm posterior to the lesion site of DMSO- and rapamycin-treated zebrafish, respectively (for details see “Materials and Methods” Section). Data represent means ± SEM of 8 optic nerves from two independent experiments. Treatment effects compared to DMSO control: ***p < 0.001; *p < 0.05 (D) Quantitative real-time PCR for green fluorescent protein (gfp) and growth associated protein 43 (gap43) in relation to glyceraldehyde-3-phosphate dehydrogenase (gapdh) in retinae isolated from zebrafish 2 days post injury that were treated either with vehicle (DMSO) or 0.2 μM rapamycin (Rap), respectively. Data represent mean ΔΔCt ± SEM of at least three different fish per experimental group. ns = non-significant (E) Representative pictures of the swimming position of a naïve zebrafish and a fish 1 day post unilateral right optic nerve crush (1 dpi), respectively. (F) Quantification of the oblique swimming position of DMSO (blue)- and rapamycin (Rap; red)-treated zebrafish at 1, 4, 11, 13, 14 and 18 days post injury (dpi). In addition, some zebrafish received rapamycin-treatment only during the first 3 days of the experiment (RD; brown). Another group was initially held in DMSO (0–3 dpi) and then transferred to rapamycin for the remainder of the experiment (DR; green). Data represent means ± SEM of at least five zebrafish per group. Treatment effects compared to DMSO control: ***p < 0.001; **p < 0.01.
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Figure 5: Compromised axon regeneration and functional recovery upon mTOR inhibition. (A,B) Maximum intensity projections (85 × 0.9 μm confocal z-sections) of wholemount optic nerves from DMSO- (A) and 0.2 μM rapamycin (Rap, B) -treated GAP43::GFP zebrafish at 2.5 days post injury (dpi). The lesion site is indicated with a dashed line, proximal is to the left. Scale bar = 200 μm. (A′,B′) Higher magnifications of the boxed areas in (A) and (B), respectively, using maximum intensity projections of 10 × 0.9 μm confocal stacks. Scale bar = 50 μm. (C) Quantification of axon profiles per mm optic nerve diameter on single z-sections at 200 and 500 μm posterior to the lesion site of DMSO- and rapamycin-treated zebrafish, respectively (for details see “Materials and Methods” Section). Data represent means ± SEM of 8 optic nerves from two independent experiments. Treatment effects compared to DMSO control: ***p < 0.001; *p < 0.05 (D) Quantitative real-time PCR for green fluorescent protein (gfp) and growth associated protein 43 (gap43) in relation to glyceraldehyde-3-phosphate dehydrogenase (gapdh) in retinae isolated from zebrafish 2 days post injury that were treated either with vehicle (DMSO) or 0.2 μM rapamycin (Rap), respectively. Data represent mean ΔΔCt ± SEM of at least three different fish per experimental group. ns = non-significant (E) Representative pictures of the swimming position of a naïve zebrafish and a fish 1 day post unilateral right optic nerve crush (1 dpi), respectively. (F) Quantification of the oblique swimming position of DMSO (blue)- and rapamycin (Rap; red)-treated zebrafish at 1, 4, 11, 13, 14 and 18 days post injury (dpi). In addition, some zebrafish received rapamycin-treatment only during the first 3 days of the experiment (RD; brown). Another group was initially held in DMSO (0–3 dpi) and then transferred to rapamycin for the remainder of the experiment (DR; green). Data represent means ± SEM of at least five zebrafish per group. Treatment effects compared to DMSO control: ***p < 0.001; **p < 0.01.

Mentions: Fish swim in a slightly oblique position (~10°) upon unilateral optic nerve injury (Figure 5E; Lindsey and Powers, 2007; Mensinger and Powers, 2007), which is gradually reversed with ongoing regeneration. Therefore, the degree of tilt can serve as a measure for functional regeneration. At various times after optic nerve injury, fish were placed into a 2.7 × 17.5 cm container with 400 ml water. After ~5 min adaptation, they were recorded on video for 1–2 min, making sure to capture at least five straight swims directly towards the camera. The videos were analyzed frame by frame and still pictures taken if the whole body of the fish was positioned straight towards the camera. The angle between the fish body position (straight line through the eyes; Figure 5E) and the horizon was then determined using ImageJ. At least seven different pictures were analyzed per fish and time point to calculate the mean divergent angle. Data are given as means ± SEM of at least five fish per group. The significance of intergroup differences was evaluated using Repeat-Measurements Two-Way ANOVA with Holm-Sidak post hoc tests (GraphPad; SigmaStat).


Active mechanistic target of rapamycin plays an ancillary rather than essential role in zebrafish CNS axon regeneration.

Diekmann H, Kalbhen P, Fischer D - Front Cell Neurosci (2015)

Compromised axon regeneration and functional recovery upon mTOR inhibition. (A,B) Maximum intensity projections (85 × 0.9 μm confocal z-sections) of wholemount optic nerves from DMSO- (A) and 0.2 μM rapamycin (Rap, B) -treated GAP43::GFP zebrafish at 2.5 days post injury (dpi). The lesion site is indicated with a dashed line, proximal is to the left. Scale bar = 200 μm. (A′,B′) Higher magnifications of the boxed areas in (A) and (B), respectively, using maximum intensity projections of 10 × 0.9 μm confocal stacks. Scale bar = 50 μm. (C) Quantification of axon profiles per mm optic nerve diameter on single z-sections at 200 and 500 μm posterior to the lesion site of DMSO- and rapamycin-treated zebrafish, respectively (for details see “Materials and Methods” Section). Data represent means ± SEM of 8 optic nerves from two independent experiments. Treatment effects compared to DMSO control: ***p < 0.001; *p < 0.05 (D) Quantitative real-time PCR for green fluorescent protein (gfp) and growth associated protein 43 (gap43) in relation to glyceraldehyde-3-phosphate dehydrogenase (gapdh) in retinae isolated from zebrafish 2 days post injury that were treated either with vehicle (DMSO) or 0.2 μM rapamycin (Rap), respectively. Data represent mean ΔΔCt ± SEM of at least three different fish per experimental group. ns = non-significant (E) Representative pictures of the swimming position of a naïve zebrafish and a fish 1 day post unilateral right optic nerve crush (1 dpi), respectively. (F) Quantification of the oblique swimming position of DMSO (blue)- and rapamycin (Rap; red)-treated zebrafish at 1, 4, 11, 13, 14 and 18 days post injury (dpi). In addition, some zebrafish received rapamycin-treatment only during the first 3 days of the experiment (RD; brown). Another group was initially held in DMSO (0–3 dpi) and then transferred to rapamycin for the remainder of the experiment (DR; green). Data represent means ± SEM of at least five zebrafish per group. Treatment effects compared to DMSO control: ***p < 0.001; **p < 0.01.
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Figure 5: Compromised axon regeneration and functional recovery upon mTOR inhibition. (A,B) Maximum intensity projections (85 × 0.9 μm confocal z-sections) of wholemount optic nerves from DMSO- (A) and 0.2 μM rapamycin (Rap, B) -treated GAP43::GFP zebrafish at 2.5 days post injury (dpi). The lesion site is indicated with a dashed line, proximal is to the left. Scale bar = 200 μm. (A′,B′) Higher magnifications of the boxed areas in (A) and (B), respectively, using maximum intensity projections of 10 × 0.9 μm confocal stacks. Scale bar = 50 μm. (C) Quantification of axon profiles per mm optic nerve diameter on single z-sections at 200 and 500 μm posterior to the lesion site of DMSO- and rapamycin-treated zebrafish, respectively (for details see “Materials and Methods” Section). Data represent means ± SEM of 8 optic nerves from two independent experiments. Treatment effects compared to DMSO control: ***p < 0.001; *p < 0.05 (D) Quantitative real-time PCR for green fluorescent protein (gfp) and growth associated protein 43 (gap43) in relation to glyceraldehyde-3-phosphate dehydrogenase (gapdh) in retinae isolated from zebrafish 2 days post injury that were treated either with vehicle (DMSO) or 0.2 μM rapamycin (Rap), respectively. Data represent mean ΔΔCt ± SEM of at least three different fish per experimental group. ns = non-significant (E) Representative pictures of the swimming position of a naïve zebrafish and a fish 1 day post unilateral right optic nerve crush (1 dpi), respectively. (F) Quantification of the oblique swimming position of DMSO (blue)- and rapamycin (Rap; red)-treated zebrafish at 1, 4, 11, 13, 14 and 18 days post injury (dpi). In addition, some zebrafish received rapamycin-treatment only during the first 3 days of the experiment (RD; brown). Another group was initially held in DMSO (0–3 dpi) and then transferred to rapamycin for the remainder of the experiment (DR; green). Data represent means ± SEM of at least five zebrafish per group. Treatment effects compared to DMSO control: ***p < 0.001; **p < 0.01.
Mentions: Fish swim in a slightly oblique position (~10°) upon unilateral optic nerve injury (Figure 5E; Lindsey and Powers, 2007; Mensinger and Powers, 2007), which is gradually reversed with ongoing regeneration. Therefore, the degree of tilt can serve as a measure for functional regeneration. At various times after optic nerve injury, fish were placed into a 2.7 × 17.5 cm container with 400 ml water. After ~5 min adaptation, they were recorded on video for 1–2 min, making sure to capture at least five straight swims directly towards the camera. The videos were analyzed frame by frame and still pictures taken if the whole body of the fish was positioned straight towards the camera. The angle between the fish body position (straight line through the eyes; Figure 5E) and the horizon was then determined using ImageJ. At least seven different pictures were analyzed per fish and time point to calculate the mean divergent angle. Data are given as means ± SEM of at least five fish per group. The significance of intergroup differences was evaluated using Repeat-Measurements Two-Way ANOVA with Holm-Sidak post hoc tests (GraphPad; SigmaStat).

Bottom Line: Remarkably, regulation of mTOR activity after optic nerve injury in zebrafish is fundamentally different compared to mammals.Moreover, inhibition of mTOR using rapamycin significantly reduced axon regeneration in vivo and compromised functional recovery after optic nerve injury.Therefore, axotomy-induced mTOR activity is involved in CNS axon regeneration in zebrafish similar to mammals, although it plays an ancillary rather than essential role in this regeneration-competent species.

View Article: PubMed Central - PubMed

Affiliation: Division of Experimental Neurology, Department of Neurology, Heinrich-Heine-University of Düsseldorf Düsseldorf, Germany.

ABSTRACT
The developmental decrease of the intrinsic regenerative ability of the mammalian central nervous system (CNS) is associated with reduced activity of mechanistic target of rapamycin (mTOR) in mature neurons such as retinal ganglion cells (RGCs). While mTOR activity is further decreased upon axonal injury, maintenance of its pre-injury level, for instance by genetic deletion of the phosphatase and tensin homolog (PTEN), markedly promotes axon regeneration in mammals. The current study now addressed the question whether active mTOR might generally play a central role in axon regeneration by analyzing its requirement in regeneration-competent zebrafish. Remarkably, regulation of mTOR activity after optic nerve injury in zebrafish is fundamentally different compared to mammals. Hardly any activity was detected in naïve RGCs, whereas it was markedly increased upon axotomy in vivo as well as in dissociated cell cultures. After a short burst, mTOR activity was quickly attenuated, which is contrary to the requirements for axon regeneration in mammals. Surprisingly, mTOR activity was not essential for axonal growth per se, but correlated with cytokine- and PTEN inhibitor-induced neurite extension in vitro. Moreover, inhibition of mTOR using rapamycin significantly reduced axon regeneration in vivo and compromised functional recovery after optic nerve injury. Therefore, axotomy-induced mTOR activity is involved in CNS axon regeneration in zebrafish similar to mammals, although it plays an ancillary rather than essential role in this regeneration-competent species.

No MeSH data available.


Related in: MedlinePlus