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Paclitaxel induces axonal microtubules polar reconfiguration and impaired organelle transport: implications for the pathogenesis of paclitaxel-induced polyneuropathy

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Abstract

In differentiated axons almost all microtubules (MTs) uniformly point their plus ends towards the axonal tip. The uniform polar pattern provides the structural substrate for efficient organelle transport along axons. It is generally believed that the mass and pattern of MTs polar orientation remain unchanged in differentiated neurons. Here we examined long-term effects of the MTs stabilizing reagent paclitaxel (taxol) over MTs polar orientation and organelle transport in cultured Aplysia neurons. Unexpectedly, we found that rather than stabilizing the MTs, paclitaxel leads to their massive polar reconfiguration, accompanied by impaired organelle transport. Washout of paclitaxel does not lead to recovery of the polar orientation indicating that the new pattern is self-maintained. Taken together the data suggest that MTs in differentiated neurons maintain the potential to be reconfigured. Such reconfiguration may serve physiological functions or lead to degeneration. In addition, our observations offer a novel mechanism that could account for the development of peripheral neuropathy in patients receiving paclitaxel as an antitumor drug.

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Acknowledgments

This study was supported by the Israel Ministry of Health (300000-4955). Part of the work was done at the Charles E. Smith Family and Prof. Joel Elkes Laboratory for Psychobiology. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. We thank Prof. Reuma Falk for statistical advice. Or Shemesh was partially supported by the Dimitris N. Chorafas foundation and by George Elias and Ada Tsur. M. E. Spira is the Levi DeViali Prof. in Neurobiology.

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Correspondence to Micha E. Spira.

Electronic supplementary material

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Movie S1. Paclitaxel induced MT polar reconfiguration. (A) EB3-GFP comet tails in control axon (before paclitaxel application), 100-200µm away from cell body. (B) The same axon, at the same location 72h after the application of 100nM paclitaxel into the bathing solution. The video contains 30 images, taken at intervals of 5.7 seconds. The frames are shown at a rate of 10/s. Scalebar: 10 µm (MPG 12.6 MB)

Movie S5. 100nM paclitaxel leads to severe impairments in SR101 labeled vesicles. (A) The video (green) depicts EB3-GFP comet tails in a neuron 48h after the onset of 100nM paclitaxel incubation, 200-300µm from cell body. And (B), the transport of SR101 labeled pinocytotic vesicles. The video comprises 30 images, taken at intervals of 5.5 seconds. Scalebar: 10 µm (MPG 12.6 MB)

Movie S6. 100nM paclitaxel impedes mitochondrial transport. (A) A buccal neuron was injected with hOACTL-GFP mRNA and imaged 24h later. 25 frames of hOACTL-GFP labeled mitochondria were taken 5.3s apart. (B) Thereafter, the neuron was incubated with 100nM paclitaxel and imaged 24h later. 25 frames were taken at an interval of 5.3s. (C) The axon 72h following paclitaxel incubation. 25 frames were taken at an interval of 5.3s. Scalebar: 10µm (MPG 7.64 MB)

Movie S7. Washout of paclitaxel does not lead to recovery of the MTs polar pattern. The video depicts the same neuron presented in Fig. 8, 48h following 100nM paclitaxel washout. (A) EB3-GFP (green) comet tails in a neuron, 200-300µm from cell body. (B) The transport of SR101 labeled organells. The video comprises 30 images, taken at intervals of 5.5 seconds. Scalebar: 10 µm (MPG 12.6 MB)

Supplementary material 1 (DOC 35 kb)

Supplementary material 2 (DOC 37 kb)

Movie S1. Paclitaxel induced MT polar reconfiguration. (A) EB3-GFP comet tails in control axon (before paclitaxel application), 100-200µm away from cell body. (B) The same axon, at the same location 72h after the application of 100nM paclitaxel into the bathing solution. The video contains 30 images, taken at intervals of 5.7 seconds. The frames are shown at a rate of 10/s. Scalebar: 10 µm (MPG 12.6 MB)

Movie S2. Incubation of a neuron with 10nM paclitaxel. (A) EB3-GFP comet tails in a control axon (before application of paclitaxel). (B) The same axon, at the same location 96h after the addition of 10nM paclitaxel to the bathing solution. The video contains 30 images, taken at intervals of 5.7 seconds. The frames are shown at a rate of 10/s. Scalebar: 10 µm (MPG 8.52 MB)

Movie S3. EB3-GFP comet tails and retrograde transport in a control neuron. (A) EB3-GFP comet tails in a control neuron (green), 200-300µm from cell body and (B) transport of SR101 labeled organells (red). The videos comprise 30 images, taken at intervals of 4.5 seconds. Scalebar: 10 µm (MPG 9.84 MB)

Movie S4. 10nM paclitaxel leads to impaired transport of SR101 labeled organells. (A) EB3-GFP (green) comet tails in a neuron 72h after the onset of 100nM paclitaxel incubation, 200-300µm from cell body. (B) The transport of SR101 labeled pinocytotic organelles (red). The video comprises 30 images, taken at intervals of 5.7 seconds. Scalebar: 10 µm (MPG 9.84 MB)

Movie S5. 100nM paclitaxel leads to severe impairments in SR101 labeled vesicles. (A) The video (green) depicts EB3-GFP comet tails in a neuron 48h after the onset of 100nM paclitaxel incubation, 200-300µm from cell body. And (B), the transport of SR101 labeled pinocytotic vesicles. The video comprises 30 images, taken at intervals of 5.5 seconds. Scalebar: 10 µm (MPG 12.6 MB)

Movie S6. 100nM paclitaxel impedes mitochondrial transport. (A) A buccal neuron was injected with hOACTL-GFP mRNA and imaged 24h later. 25 frames of hOACTL-GFP labeled mitochondria were taken 5.3s apart. (B) Thereafter, the neuron was incubated with 100nM paclitaxel and imaged 24h later. 25 frames were taken at an interval of 5.3s. (C) The axon 72h following paclitaxel incubation. 25 frames were taken at an interval of 5.3s. Scalebar: 10µm (MPG 7.64 MB)

Movie S7. Washout of paclitaxel does not lead to recovery of the MTs polar pattern. The video depicts the same neuron presented in Fig. 8, 48h following 100nM paclitaxel washout. (A) EB3-GFP (green) comet tails in a neuron, 200-300µm from cell body. (B) The transport of SR101 labeled organells. The video comprises 30 images, taken at intervals of 5.5 seconds. Scalebar: 10 µm (MPG 12.6 MB)

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Shemesh, O.A., Spira, M.E. Paclitaxel induces axonal microtubules polar reconfiguration and impaired organelle transport: implications for the pathogenesis of paclitaxel-induced polyneuropathy. Acta Neuropathol 119, 235–248 (2010) doi:10.1007/s00401-009-0586-0

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Keywords

  • Paclitaxel
  • Microtubules
  • Plus ends binding proteins
  • Axoplasmic transport
  • Peripheral neuropathy
  • Aplysia