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Schwann Cells in Paclitaxel-Induced Sensory Peripheral Neuropathy
Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Pharmaceutical Biosciences.
2019 (English)Independent thesis Advanced level (degree of Master (One Year)), 20 credits / 30 HE creditsStudent thesis
Abstract [en]

Introduction: Sensory peripheral neuropathy (SPN) is an adverse event associated with paclitaxel treatment. It causes dose reductions and reduced patient quality of life, and there is currently no available treatment. In the peripheral nervous system, axons are myelinated by Schwann cells (SC). SCs have a major function in the healthy nervous system and in nerve repair, raising the possibility that SC impairment could be one of the causes of paclitaxel-induced SPN. A previous experiment in the Kroetz laboratory showed that paclitaxel treatment induced morphology changes in SCs, possibly caused by activation of RhoA signaling. Recent studies also suggest that endocytosis is of importance for SC function. It has been shown that mutations associated with the demyelinating Charcot-Marie-Tooth disease also cause impaired SC endocytosis. Furthermore, it has been demonstrated that the low-density lipoprotein receptor-related protein-1 (LRP-1), an endocytic receptor, is of importance for clearing of degraded myelin in the central nervous system.

Aim: The aim of this study was to increase the understanding of the role of SCs in paclitaxel- induced SPN. First, the cell line used for experiments was validated. Thereafter, the possible activating effect of paclitaxel on RhoA signaling was investigated. Finally, the potential inhibitory effect of paclitaxel on SC endocytosis was explored.

Materials and Methods: The experiments within the project were performed using the rat schwannoma cell line RT4-D6P2T (RT4). Validation of the cell line was performed by measurement of five known SC markers using three different methods. The mRNA expression of the markers was determined using quantitative polymerase chain reaction (qPCR), while the protein expression was measured using Western blot and immunocytochemistry. The effect of paclitaxel on RhoA signaling was determined by measuring myosin light chain (MLC) phosphorylation using Western blot. Transferrin receptor endocytosis was used as a model for measurement of endocytic activity. Alexa Fluor 647-labeled transferrin was used to enable visualization of possible paclitaxel-induced changes.

Results: qPCR results showed that S100, p75, LRP-1 and galectin-3 were all highly expressed in the RT4 cell line, while myelin basic protein (MBP) had almost undetectable expression values. The qPCR also revealed that the expression of the two reference genes b-actin and GAPDH was unstable and appeared to decrease after paclitaxel treatment. Western blot indicated a dose-dependent increase in expression of p75, LRP-1 and galectin-3, while the expression of S100 was more constant. Immunocytochemistry results showed that p75, galectin-3, LRP-1 and S100 were all expressed in the RT4 cells, both in the negative control and after paclitaxel treatment. Paclitaxel activation of RhoA signaling could not be shown or ruled out. No signal for MLC phosphorylation was detected after paclitaxel treatment and only a weak signal was detected after treatment with the positive control lysophosphatidic acid. Paclitaxel treatment appeared to cause reduced transferrin receptor endocytosis. However, chlorpromazine, the positive control for inhibition of endocytosis, did not cause reduced receptor internalization.

Conclusion: The RT4 cell line is partially similar to primary SCs in terms of marker gene expression and can therefore be used to study the effect of paclitaxel on SCs. Due to technical issues, paclitaxel activation of RhoA signaling could not be confirmed or ruled out. The assay therefore needs to be optimized before any conclusion can be made. Experiments in this project indicate that paclitaxel has an inhibitory effect on SC endocytosis. This finding has to confirmed by further studies. Additionally, the effect of paclitaxel on transferrin receptor recycling should be determined.

Place, publisher, year, edition, pages
2019.
Keywords [en]
Schwann cell, paclitaxel, neuropathy
National Category
Pharmaceutical Sciences
Identifiers
URN: urn:nbn:se:uu:diva-377320OAI: oai:DiVA.org:uu-377320DiVA, id: diva2:1289723
External cooperation
Kroetz Laboratory, University of California San Francisco
Subject / course
Pharmacokinetics
Educational program
Master of Science Programme in Pharmacy
Supervisors
Examiners
Available from: 2019-02-21 Created: 2019-02-18 Last updated: 2019-02-21Bibliographically approved

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