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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

Correlation of mTOR activity with CNTF- and PTEN inhibitor-induced neurite growth of cultured RGCs. (A) Representative pictures of cultured retinal ganglion cells (RGCs) stained with acetylated tubulin antibody at 4 days in culture and exposure to vehicle (con) and ciliary neurotrophic factor (CNTF, 1 ng/ml), respectively. Scale bar = 50 μm. (B) Quantification of average RGC neurite length at 4 days in culture after treatment with either vehicle control (−), rapamycin (Rap; 10 nM), PTEN inhibitor bpV(phen) (10 nM), or bpV(phen) + Rap. Data represent means ± SEM of at least two independent experiments, each with three repeats. Treatment effects: p < 0.001.
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Figure 3: Correlation of mTOR activity with CNTF- and PTEN inhibitor-induced neurite growth of cultured RGCs. (A) Representative pictures of cultured retinal ganglion cells (RGCs) stained with acetylated tubulin antibody at 4 days in culture and exposure to vehicle (con) and ciliary neurotrophic factor (CNTF, 1 ng/ml), respectively. Scale bar = 50 μm. (B) Quantification of average RGC neurite length at 4 days in culture after treatment with either vehicle control (−), rapamycin (Rap; 10 nM), PTEN inhibitor bpV(phen) (10 nM), or bpV(phen) + Rap. Data represent means ± SEM of at least two independent experiments, each with three repeats. Treatment effects: p < 0.001.

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)

Correlation of mTOR activity with CNTF- and PTEN inhibitor-induced neurite growth of cultured RGCs. (A) Representative pictures of cultured retinal ganglion cells (RGCs) stained with acetylated tubulin antibody at 4 days in culture and exposure to vehicle (con) and ciliary neurotrophic factor (CNTF, 1 ng/ml), respectively. Scale bar = 50 μm. (B) Quantification of average RGC neurite length at 4 days in culture after treatment with either vehicle control (−), rapamycin (Rap; 10 nM), PTEN inhibitor bpV(phen) (10 nM), or bpV(phen) + Rap. Data represent means ± SEM of at least two independent experiments, each with three repeats. Treatment effects: p < 0.001.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC4493654&req=5

Figure 3: Correlation of mTOR activity with CNTF- and PTEN inhibitor-induced neurite growth of cultured RGCs. (A) Representative pictures of cultured retinal ganglion cells (RGCs) stained with acetylated tubulin antibody at 4 days in culture and exposure to vehicle (con) and ciliary neurotrophic factor (CNTF, 1 ng/ml), respectively. Scale bar = 50 μm. (B) Quantification of average RGC neurite length at 4 days in culture after treatment with either vehicle control (−), rapamycin (Rap; 10 nM), PTEN inhibitor bpV(phen) (10 nM), or bpV(phen) + Rap. Data represent means ± SEM of at least two independent experiments, each with three repeats. Treatment effects: p < 0.001.
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