Key Event Title
|Level of Biological Organization|
Key Event Components
Key Event Overview
AOPs Including This Key Event
|AOP Name||Role of event in AOP|
|Microtubule interacting drugs lead to peripheral neuropathy||KeyEvent|
Key Event Description
The cytoskeleton plays an important role in neurons as it is required for the typical neuronal architecture of one long process, the axon, and several shorter dendrites.  Furthermore, the intact cytoskeleton is also of high importance as it is needed for processes like axonal transport. As axons lack the machinery to synthesize proteins, all necessary proteins have to be transported from the cell body to the periphery. Microtubules which are a basic element of the cytoskeleton play an important role in axonal transport and the maintenance of neurons.  They are highly dynamic and polarized structures with a stable minus end and a dynamic plus end. In axons, the plus end is directed away from the soma.  Microtubules serve as molecular tracks in neurons to ensure the transport of cargoes to different parts of the cell as well as the clearance of damaged cell organelles. The kinesins are microtubule-based molecular motors and are necessary for the anterograde transport of materials needed for maintenance of axons and synapses. [3, 4] Retrograde transport of degradation products from the axon/synapse back to the cell body is crucial for neuronal maintenance and survival as well.  Retrograde transport is carried out by dynein-motorproteins. 
How It Is Measured or Detected
- Vesicle motility assay: Axoplasm from squid giant axons is isolated and kept in axoplasm buffer. Preparations are analysed using a Zeiss Axiomat and organelle velocities are measured either in an automated process or by matching calibrated cursor movements to the speed of moving vesicles in agreement of two observers. [7-9]
- Kinesin-driven microtubule gliding assay: Slide chambers are covered with kinesins which adhere e.g. to specific antibodies on the glass slides. Rhodamine-labelled tubulin and unlabelled tubulin are mixed and assembled to microtubule structures. Microtubules are applied to the chamber and the rhodamine fluorescence is visualized to evaluate microtubule gliding. Microtubule-bodies are located and tracked to collect data on gliding velocity, trajectory curvature and microtubule length. [7, 10]
- Horseradish peroxidase (HRP) microinjection: HRP is injected into dorsal root ganglia neurons and visualized by 3,3’-diaminobenzidine. Microscope recordings of the neurons showing the transport of HRP are evaluated and the transport length is measured. 
- Mitochondrial trafficking: Cells are incubated with drug or DMSO solution and afterwards mitochondria are labelled with MitoTracker Green FM. Cells are kept in a live cell chamber and imaged in regular intervals. The time-lapse is used to track mitochondrial movement in neurites. 
- Axonal transport in mouse sciatic nerve: The drug is administered to mice intravenously. Mice are anesthetized and the left sciatic nerve is exposed and ligated at two points. After 24h, the ligated sciatic nerves are dissected and segments from proximal and distal sides of the ligation are collected, homogenized and analysed by Western blot. 
Domain of Applicability
1. Baas, P.W., et al., Stability properties of neuronal microtubules. Cytoskeleton (Hoboken), 2016. 73(9): p. 442-60.
2. Hirokawa, N., Axonal transport and the cytoskeleton. Current Opinion in Neurobiology, 1993. 3(5): p. 724-731.
3. Leopold, P.L., et al., Association of kinesin with characterized membrane-bounded organelles. Cell Motility and the Cytoskeleton, 1992. 23(1): p. 19-33.
4. Elluru, R.G., G.S. Bloom, and S.T. Brady, Fast axonal transport of kinesin in the rat visual system: functionality of kinesin heavy chain isoforms. Molecular Biology of the Cell, 1995. 6(1): p. 21-40.
5. Delcroix, J.-D., et al., Trafficking the NGF signal: implications for normal and degenerating neurons, in Progress in Brain Research. 2004, Elsevier. p. 1-23.
6. Susalka, S.J. and K.K. Pfister, Cytoplasmic dynein subunit heterogeneity: implications for axonal transport. Journal of Neurocytology, 2000. 29(11): p. 819-829.
7. LaPointe, N.E., et al., Effects of eribulin, vincristine, paclitaxel and ixabepilone on fast axonal transport and kinesin-1 driven microtubule gliding: implications for chemotherapy-induced peripheral neuropathy. Neurotoxicology, 2013. 37: p. 231-9.
8. Morfini, G., et al., Tau binding to microtubules does not directly affect microtubule‐based vesicle motility. Journal of Neuroscience Research, 2007. 85(12): p. 2620-2630.
9. Morfini, G., et al., JNK mediates pathogenic effects of polyglutamine-expanded androgen receptor on fast axonal transport. Nature Neuroscience, 2006. 9: p. 907.
10. Peck, A., et al., Tau isoform‐specific modulation of kinesin‐driven microtubule gliding rates and trajectories as determined with tau‐stabilized microtubules. Cytoskeleton, 2011. 68(1): p. 44-55.
11. Theiss, C. and K. Meller, Taxol impairs anterograde axonal transport of microinjected horseradish peroxidase in dorsal root ganglia neurons in vitro. Cell Tissue Res, 2000. 299(2): p. 213-24.
12. Smith, J.A., et al., Structural Basis for Induction of Peripheral Neuropathy by Microtubule-Targeting Cancer Drugs. Cancer Research, 2016. 76(17): p. 5115-5123.