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. Author manuscript; available in PMC: 2021 Sep 1.
Published in final edited form as: J Neurochem. 2020 Mar 13;154(6):662–672. doi: 10.1111/jnc.14985

Role of 1-Deoxysphingolipids in docetaxel neurotoxicity

Katrin Anne Becker 1,*, Anne-Kathrin Uerschels 2,*, Laura Goins 3, Suzanne Doolen 4, Kristen Jean McQuerry 5, Jacek Bielawski 6, Ulrich Sure 2, Erhard Bieberich 3, Bradley K Taylor 4, Erich Gulbins 1, Stefka D Spassieva 3,#
PMCID: PMC7426245  NIHMSID: NIHMS1571417  PMID: 32058598

Abstract

A major dose-limiting side effect of docetaxel chemotherapy is peripheral neuropathy. Patients’ symptoms include pain, numbness, tingling and burning sensations, and motor weakness in the extremities. The molecular mechanism is currently not understood, and there are no treatments available. Previously, we have shown an association between neuropathy symptoms of patients treated with paclitaxel and the plasma levels of neurotoxic sphingolipids, the 1-deoxysphingolipids (1-deoxySL) (Kramer et al, FASEB J, 2015). 1-deoxySL are produced when the first enzyme of the sphingolipid biosynthetic pathway, serine palmitoyltransferase (SPT), uses L-alanine as a substrate instead of its canonical amino acid substrate, L-serine. In the current investigation, we tested whether 1-deoxySL accumulate in the nervous system following systemic docetaxel treatment in mice. In dorsal root ganglia (DRG), we observed that docetaxel (45 mg/kg cumulative dose) significantly elevated the levels of 1-deoxySL and L-serine-derived ceramides, but not sphingosine-1-phosphate (S1P). S1P is a bioactive sphingolipid and a ligand for specific G-protein coupled receptors. In the sciatic nerve, docetaxel decreased 1-deoxySL and ceramides. Moreover, we show that in primary DRG cultures, 1-deoxysphingosine produced neurite swellings that could be reversed with S1P. Our results demonstrate that docetaxel chemotherapy up-regulates sphingolipid metabolism in sensory neurons, leading to the accumulation of neurotoxic 1-deoxySLs. We suggest that the neurotoxic effects of 1-deoxySL on axons can be reversed with S1P.

Keywords: 1-deoxysphingolipids, Serine palmitoyltransferase, Sphingosine-1-phosphate, ceramide, Docetaxel-induced peripheral neuropathy

Graphical Abstract

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In the dorsal root ganglia of mice treated with docetaxel, a peripheral neuropathy-inducing chemotherapeutic agent, we discovered dysregulation of sphingolipid metabolism. Specifically, the levels of neurotoxic 1-deoxysphingolipids were increased, along with dihydroceramides and ceramides. The levels of the bioactive sphingolipid metabolite, sphingosine-1-phosphate, were not changed, however. Moreover, our in vitro data with primary dorsal root ganglia neurons show that treatment with 1-deoxysphingosine resulted in neurite swellings and abnormalities, which were reversed by co-treatment with sphingosine-1-phospahte. We suggest that exploring sphingosine-1-phosphate signaling can identify targets to address taxane-induced neuropathy.

Introduction

Taxanes, i.e., paclitaxel and docetaxel, are chemotherapy drugs that are widely used to treat a variety of cancers (Ghersi et al. 2015, Zaheed et al. 2019, Marchetti et al. 2018, Chin et al. 2018). However, taxane-induced peripheral neuropathy is a commonly occurring side effect of taxane chemotherapy (Song et al. 2017, Velasco & Bruna 2015, Rowinsky et al. 1993). Symptoms include tingling, numbness, shooting pain, and in some cases, burning sensations in the hands and feet. If severe, these adverse effects force dose reductions or even termination of treatment, leading to loss of efficacy of taxane chemotherapy (Bhatnagar et al. 2014). In addition, taxane-induced peripheral neuropathy persists for months or even years after treatment cessation, which considerably diminishes patients’ quality of life even after remission (Molassiotis et al. 2019). In our previous study in patients receiving standard paclitaxel chemotherapy, we found that the incidence and severity of peripheral neuropathy symptoms associate with the level of certain 1-deoxySL species (Kramer et al. 2015).

1-DeoxySL are a minor sphingolipid class, which is produced when the first enzyme of the sphingolipid biosynthetic pathway, SPT, uses L-alanine for its amino acid substrate rather than its canonical substrate, L-serine (Figure 1) (Gable et al. 2010, Penno et al. 2010, Zitomer et al. 2009). As a result, the L-alanine-derived 1-deoxySL do not possess the important functional hydroxyl group at the C1 position as opposed to their L-serine-derived counterparts. In L-serine-derived sphingolipids, this hydroxyl group is used for the attachment of the headgroups of complex sphingolipids, as well as to activate sphingoid bases, such as sphingosine and sphinganine, to their phosphate derivatives, S1P and sphinganine-1-phosphate, respectively (Figure 1) (reviewed in (Merrill 2011)). S1P is a signaling molecule in its own right, implicated in regulating cell survival, cell proliferation, and migration (reviewed in (Maceyka et al. 2012)). In the nervous system, S1P has been shown to differentially regulate neurite retraction and extension through engagement of different S1P receptors, S1PRs (Toman et al. 2004). There are five S1PRs expressed in DRG neurons (Mair et al. 2011, Zhang et al. 2006, Kays et al. 2012). Notably, a very recent study in an animal model of paclitaxel neuropathy has shown that modulators of S1PR subtype 1 (S1PR1) such as FTY720 can reduce neuropathic pain symptoms if administered together with paclitaxel (Janes et al. 2014).

Figure 1. Sphingolipid pathway.

Figure 1.

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A/A schematic representation of the sphingolipid pathway at the level of sphingoid bases and ceramides. 1-DeoxySL are produced when serine palmitoyl transferase uses L-alanine as the amino acid substrate. 1-DeoxySL species (derived from L-alanine) do not have an OH group at position C1, as do the sphingolipid species derived from L-serine.

S1P and sphinganine-1-phosphate are the two known sphingolipid metabolites for catabolic exit from the sphingolipid pathway, which is not available for 1-deoxySL. When 1-deoxySL are produced in excess, they accumulate; however, currently it is not clear how they are degraded. A recent study suggested that the less specific cytochrome P450 pathway could slowly metabolize 1-deoxysphingosine with 14Z double bond (Alecu et al. 2017a). The neurotoxicity of 1-deoxySL was investigated in vitro in several recent studies, implicating them in causing mitochondrial dysfunction or cytoskeletal impairments (Guntert et al. 2016, Jun et al. 2015, Alecu et al. 2017b, Wilson et al. 2018). 1-DeoxySL have also been shown in vitro to have different biophysical properties than the L-serine derived ceramides, including reduced miscibility with sphingomyelin in bilayers (Jimenez-Rojo et al. 2014). It is likely that the distinct biophysical properties of 1-deoxySL and regular ceramides result in different effects on the biological membranes. Therefore, if 1-deoxySL are produced above physiological levels their effects on cellular membranes could be disruptive.

Plasma 1-deoxySL are representative of liver metabolism and our clinical data does not provide direct evidence whether systemic taxane treatment has an effect on 1-deoxySL metabolism in the peripheral nervous system (PNS). To address this question, we explore a docetaxel mouse model of chemotherapy-induced neuropathy to determine the levels of SPT and 1-deoxySLs in the PNS. We also used in vitro primary DRG neuronal cultures to further study 1-deoxySL toxicity and its interaction with S1P signaling.

Materials and Methods:

Docetaxel treatment of mice.

Female C57BL/6JOlaHsd mice were bred and housed in the animal facility at the University of Duisburg-Essen, Germany. The strain originally derived from Harlan Laboratories (now Envigo; order code: 057, RRID #5657800). We used female mice with an age of at least 8 weeks and a weight ranging between 19g and 22g at the start of the experiments. Animals were housed in type III cages (Makrolon, top-filter) with 2 to 5 companions. Mice had ad libitum access to food and water and were kept on a 12 h/12 h light/dark cycle. Each mouse had an ear-tag and an internal ID. Mice were arbitrarily assigned to the experimental groups. Litter mates were separated in different experimental groups. Each experimental group was assigned with 3 or 4 mice. No sample calculation was performed. Three or four mice per treatment group were chosen based on the previously published mass spectrometry measurements of ceramides and 1-deoxySL in mice nervous tissues, which used the same numbers per treatment group and provided statistically significant differences between the groups (Spassieva et al. 2016). Mice were injected intraperitoneally (ip.) with docetaxel (15 mg/kg; from Taxotere®) (Sanofi Aventis Deutschland GmbH, Frankfurt am Main, Germany, license #EU1–95-002–005). Preliminary studies (9 mice) indicated that just one docetaxel injection did not change 1-deoxySL levels. To better mimic clinical chemotherapy protocols, we treated mice with docetaxel three times with an interval of four weeks between each injection (45 mg/kg cumulative dose) (Fig. 2). The total number of experimental animals was 22, including nine in the preliminary studies and three controls. The exclusion criteria included weight loss, apathy, or signs of distress. The experimental animals did not show any signs of suffering after docetaxel injections and no mice were excluded from the study. Medications to alleviate potential pain were not considered because the study investigated taxane-induced neuropathy and the use of pain medications could potentially alter the experimental readouts. Mice were sacrificed (CO2 euthanasia without anesthesia) and the DRG, the sciatic nerve, and the spinal cord were isolated from an untreated control group (3 mice) or from three docetaxel treatment groups; i.e., 2h (3 mice), 6h (3 mice), or 24h (4 mice) after the third docetaxel injections. For immunohistochemistry analyses, the samples were fixed in paraformaldehyde (PFA) (Carl Roth, Karlsruhe, Germany, cat #0335.3) (see Immunohistochemistry section of materials and methods) or snap frozen in liquid N2 for lipid analyses. Animal studies were performed in accordance with the guidelines of the German Animal Protection Law and approved by the Landesamt für Natur, Umwelt und Verbraucherschutz, Düsseldorf, Germany (Az.: 8.87–50.10.34.08.081).

Figure 2. Schematic representation of the docetaxel treatment and tissue collections time line.

Figure 2.

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C57BL/6JOlaHsd female mice, three (2h and 6h tissue collection) or four (24h tissue collection) per group, were used in the experiment. The time line is not in scale.

Lipid extraction and mass spectrometry analyses of sphingolipids:

Spinal cord samples were first homogenized with Benchtop Polytron homogenizer PT2100 (Kinematica, Bohemia, NY) in 20mM HEPES buffer pH 7.4 (Sigma Aldrich, St. Louis, MO, cat #H3375). Protein concentration was determined by Bradford assay (BioRad, Hercules, CA, cat #5000006). Homogenate equivalent of 1mg total protein from each sample was fortified with internal standards (N-palmitoyl-D-erythro-C13 sphingosine, and N-docosanoyl-D-erythro-C13 sphingosine, 50nmol each) and used for lipid extraction. Next, samples were extracted two times with 2ml ethyl acetate/isopropanol/water (60/30/10, v/v/v). Lipid extracts were separated by a 10 min centrifugation at 5000xg and 10°C. Both lipid extracts were combined, dried under a stream of N2, and re-dissolved in 150µl of 1mM NH4COOH (Sigma Aldrich, cat #516961) in 0.2% HCOOH (Sigma Aldrich, cat #543804) in CH3OH (Sigma Aldrich, cat #34860) before subjected to liquid chromatography tandem mass spectrometry analyses (LC/MS/MS) of 1-deoxySL and ceramides. DRG and sciatic nerve samples were fortified with the internal standards (see above) and extracted four times with 1ml of the extraction solvent (see above) by vortexing for 1min and sonicating 10min in a water bath sonicator, followed by centrifugation for 10min at 5000xg and 10°C. Samples were kept on ice between vortexing and sonication. The four extracts were combined, dried under a stream of N2, and re-dissolved in 150µl of 1mM NH4COOH in 0.2% HCOOH in CH3OH before subjected to LC/MS/MS. The LC/MS/MS analyses were performed as described previously (Kramer et al. 2015). Mass spectrometry results from the spinal cord samples were normalized to total protein content and from DRG and sciatic nerves to total lipid phosphate content as described before (Van Veldhoven & Bell 1988). For lipid extractions and mass spectrometry analyses of lipids the samples were assigned de-identifying numbers. The experimenters performing the lipid analyses were not aware of the experimental groups.

Immunohistochemistry:

DRG were fixed for 40h with 4% PFA (Sigma-Aldrich, Steinheim, Germany) in phosphate buffered saline (PBS: 137mM NaCl, 2.7mM KCl, 7mM CaCl2, 0.8mM MgSO4, 1.4mM KH2PO4, and 6.5mM Na2HPO4, pH 7.3), dehydrated, and embedded in paraffin. Subsequently, paraffin-embedded slides were sequentially treated with 100% xylene (twice for 10min each), 100% ethanol (twice for 10min each), 95%, 70%, and 50% ethanol for 5min each, and in PBS for 10min. Next, the slides were blocked in 200µL of 3% ovalbumin (Thermo Fisher, Waltham, MA, Cat #BP25355) in PBS (Thermo Fisher, Cat #AM9625) at 37°C for 1h; incubated with mouse anti-LCB1 antibody (BD Biosciences Cat# 611305, RRID:AB_398831) in 0.1% ovalbumin in PBS overnight at 4°C; and then washed twice with PBS for 10min each. Next, the slides were incubated with Cy3 AffiniPure Donkey Anti-Mouse IgG (H+L) (Jackson ImmunoResearch Labs Cat# 715–165-151, RRID:AB_2315777) in 0.1% ovalbumin in PBS for 2h at 37°C; washed twice with 0.1% TritonX-100 (Sigma Aldrich, Cat #X100) in PBS for 10min each, followed by PBS twice for 5min each. Finally, slides were incubated with NeuroTrace 640/660 Deep-Red Fluorescent Nissl Stain ( Thermo Fisher Scientific Cat# N21483, RRID:AB_2572212) for 60 min at 24°C; washed twice with 0.1% TritonX-100 in PBS for 10min each, followed by PBS twice for 5min each. Histology mounting medium was applied to each slide using Fluoroshield with DAPI, (Sigma Aldrich, Cat #F6057). Images were captured on Nikon Ti2E inverted fluorescence microscope (Nikon Instruments Inc., Melville, NY) and analyzed using Nikon Elements Imaging Software. The experimenter analyzing the images was unaware of the experimental groups and was different from the experimenter who processed the slides and captured the images.

Mouse primary DRG neurons isolation and in vitro culture:

DRG neurons were isolated from spinal segments L3, L4, and L5 of 10-week-old C57BL/6 mice ( IMSR Cat# JAX:000664, RRID:IMSR_JAX:000664) as previously described (Woolf et al. 2008). Briefly, under isofluorane (5% induction) anesthesia, mice were euthanized by exsanguination/cardiac perfusion. Subsequently, after laminectomy, DRGs were harvested and cut into small pieces, which were transferred to micro centrifuge tubes containing Hank’s balanced salt solution (HBSS) w/o Ca/Mg (Thermo Fisher, Cat # 14185052). The DRG neurons were dissociated into single cells by digestions in enzyme solutions, pelleted, triturated, and re-pelleted. DRG neurons were cultured for 48h at 37°C and 5% CO2 in Ham’s F12 culture media (VWR, Radnor, PA, Cat # 45000–358), supplemented with 10% fetal bovine serum (Thermo Fisher, Cat #16000044) and 1% penicillin/streptomycin (Thermo Fisher, cat #15140122) on 6-well glass bottom plates (Mattek, Ashland, MA, cat #P12G-0–10-F) coated with poly-L-ornithine (Sigma Aldrich, cat #P4957) and laminin (Thermo Fisher, cat #23017015). The DRG neuron isolation was performed three times, each time the DRGs from one mouse were used, three mice in total were used in the experiments. Animal studies were approved by the Institutional Animal Care and Use Committee at the University of Kentucky, Lexington, KY, USA (protocol # 2018–2952).

Lipid treatment and live imaging of cultured DRG neurons:

48h after the DRG neurons were isolated, the culture media was replaced with Ham’s F12 media without phenol red (Caisson Labs, Smithfield, UT, Cat #HFL05), supplemented with 10% dialyzed (lipid-reduced) fetal bovine serum (Thermo Fisher, Cat #A3382001) and incubated with 75nM SiR Actin (Cytoskeleton, Inc., Denver, CO, Cat# CY-SC001). Subsequently, 6h after SirActin was added to the DRG neuronal culture, lipids were added as Huzzah® conjugates (Avanti Polar Lipids, Alabaster, AL, Cat# 360000P), and individual neurons were live-imaged on Nikon Ti2E inverted fluorescence microscope with motorized stage (Nikon Instruments Inc., Melville, NY) for 6h. Images were analyzed using Nikon Elements Imaging Software. The experimenter analyzing the images of the DRG neurons was unaware of the experimental treatment applied and was different from the experimenter who treated the DRG neurons and captured the images.

Statistical Analyses:

All statistical analyses were completed in SAS 9.4 (SAS Institute Inc., Cary, NC, USA). For each outcome, a Wilcoxon rank-sum test was performed to test for differences among Wilcoxon scores (ranks among the observations) between the control group and each treatment group. Since small sample sizes analyzed, non-parametric tests were utilized, and therefore, normality and outlier tests were not conducted. Significance was set at p<0.05.

Results

1-DeoxySL are upregulated in the DRG of mice treated with docetaxel

We previously reported that taxane-induced neuropathy is associated with increased plasma levels of specific 1-deoxySL (Kramer et al. 2015). Clinical investigations of taxane-induced neuropathy such as ours, while providing valuable information, are limited and cannot deliver direct biochemical or lipid data from the PNS and/or the spinal cord. We used a mouse model to test whether treatment with a taxane (docetaxel) can affect the 1-deoxySL levels in the PNS and/or in the spinal cord. Specifically, female C57BL/6 mice were intraperitoneally injected with docetaxel three times (45mg/kg cumulative dose) with 4 weeks intervals between each injection. DRG, sciatic nerve, and spinal cord samples were collected from animals sacrificed at 2h, 6h, or 24h after the third injection or from a control group. Lipids were extracted from the collected samples and analyzed by targeted LC/MS/MS (see Methods). Supplemental tables 1–2 and Figure 3 illustrate that, several 1-deoxydihydroceramides (C18, C20, and C22) (Suppl. Table 1; Fig. 3A, B, and C), 1-deoxyceramides (C22 and C24) (Suppl. Table 2; Fig. 3D and E), and 1-deoxysphingosine (Suppl. Table 2; Fig. 3F) were significantly elevated at 6h and/or at 24h after the 3rd docetaxel injection, as compared to control mice that did not receive docetaxel. These data further demonstrate that repeated systemic treatment with a taxane can upregulate the levels of 1-deoxySL not only in the plasma, as it was published in our previous study with breast cancer patients (Kramer et al. 2015), but also in the DRG.

Figure 3. 1-DeoxySL species upregulated in the DRG of mice treated with docetaxel.

Figure 3.

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Mice (C57BL/6OlaHsd) were intraperitoneally injected three times (with four-week intervals) with docetaxel (cumulative dose 45mg/kg) or vehicle. DRG were harvested 6h and 24h after the third injection. Lipids were extracted from the isolated DRG and subjected to targeted mass spectrometry analyses of 1-deoxySL. Each treatment group consisted of three (control and 6h) or four (24h) mice. The statistical analyses are presented in Suppl. Tables 1 and 2. A/ C18 1-deoxydihydroceramide; B/ C20 1-deoxydihydroceramide; C/ C22 1-deoxydihydroceramide; D/ C22 1-deoxyceramide; E/ C24 1-deoxyceramide; and F/ 1-deoxysphingosine. The box plots were generated in Sigma plot 14.0 (Systat Software in San Jose, CA). For the treatment groups of three samples each line represents the actual lipid measurement. For the treatment group with four samples, the whiskers represent the higher and the lower value of the lipid measurement.

Moreover, we did not observe significant changes in the 1-deoxySL in the spinal cord (Suppl. Table 3). These results corroborate previous research, which show that taxanes, in this case docetaxel, are not efficiently transported through the blood brain barrier and their effects are therefore limited to the periphery (Eiseman et al. 1994, Kemper et al. 2004, Seewaldt et al. 1994).

SPTLC1 and L-serine-derived dihydroceramides and ceramides were upregulated in the DRG of mice treated with docetaxel

Our previous research in cells revealed that higher levels of 1-deoxySL are caused by upregulation of the two major subunits of SPT, SPTLC1 and SPTLC2 (Kramer et al. 2015). Next, we tested whether SPTLC1 subunit was upregulated in the DRG of mice treated with docetaxel. We used SPTLC1 subunit-specific immunofluorescence in combination with DRG neuron-visualizing Nissl fluorescence labeling. Our results with DRG samples isolated from mice treated with three docetaxel injections (Fig. 4) showed that treatment compared to control resulted in increased immunoreactivity with SPTLC1 specific antibody in the Nissl-labeled neurons. We used DRG samples collected from the docetaxel treatment groups that showed increase in the levels of 1-deoxySL; i.e. 6h and 24h after the third injection. These data are in agreement with our previous results with paclitaxel in cells, which showed that taxane treatment leads to upregulation of SPT (Kramer et al. 2015) and that in the DRG there is an upregulation of 1-deoxySL when mice were treated with docetaxel (Fig. 3).

Figure 4. SPTLC1 is upregulated in the DRG of mice treated with docetaxel.

Figure 4.

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Mice (C57BL/6OlaHsd) were intraperitoneally injected three times (with four-week intervals) with docetaxel (cumulative dose 45mg/kg). DRG were harvested 6h and 24h after the third injection of docetaxel or from the control group. Immunohistochemistry was performed on paraffin-fixed DRG slides and imaged by Nikon epifluorescence microscope. Nissl staining – green; SPTLC1 – red; DAPI- blue. A/ representative image of DRG isolated from control mouse; B/ DRG representative image of DRG isolated 6h after the 3rd injection from mouse treated with docetaxel; C/ representative image of DRG isolated 24h after the 3rd injection from mouse treated with docetaxel. D/ Nikon Elements analysis software was used to quantify the levels of fluorescence of Nissl and SPTLC1 labeling. All images were taken with the same microscope settings, and the same parameters of the software were used to analyze all of the images. DRG slides from three individual mice from each group were analyzed blindly as described in the material and methods. Technical repeats: three slides were used from each individual mouse/DRG with at least three regions of interest per slide. Statistical analyses (SAS Institute Inc., Cary, NC, USA): Ratio SPTLC1: Nissl, a Wilcoxon rank-sum test was performed to test for differences among Wilcoxon scores (ranks among the observations) between the control group and each treatment group. Significance was set at p<0.05. Control group compared to 6h after the 3rd docetaxel injection group p=0.0495. Control group compared to 24h after the 3rd docetaxel injection group p=0.0495.

Upregulation of SPT is likely to result in the upregulation of not only L-alanine-derived 1-deoxySL, but also in the upregulation of the canonical L-serine-derived sphingolipids (Fig. 1). To test if docetaxel treatment affected the levels of L-serine-derived sphingolipids in the DRG, we performed LC/MS/MS analyses of dihydroceramides and ceramides. Dihydroceramides and ceramides are the hydrophobic backbones of sphingolipids (Merrill 2011). Our data (Suppl. Tables 4 and 5) show that the majority of dihydroceramide and ceramide species are upregulated in the DRG of the 6h and 24h treatment groups after the 3rd docetaxel i.p. injection compared to the control group. These results show that the 1-deoxySL and SPT are upregulated at the same time points, in which the canonical ceramides were upregulated as well, i.e., 6h and 24h after the 3rd docetaxel injection (Suppl. Tables 1, 2, 4, and 5). In the spinal cord, however, the levels of dihydroceramides and ceramides remained unaffected (Suppl. Tables 6 and 7). Taken together our data suggest that systemic treatment with docetaxel upregulates sphingolipid metabolism in the DRG, but not in the spinal cord.

In sciatic nerve, docetaxel treatment leads to a decrease of ceramide and 1-deoxySL levels

In addition to the DRG samples, we collected sciatic nerve samples from the docetaxel-treated and control mice. We used the sciatic nerve samples to extract the lipids and measured the levels of 1-deoxySL and L-serine-derived canonical sphingoid bases and ceramides. Considering our DRG data, (see above), the results from the sciatic nerve samples were somewhat unexpected. In the docetaxel treatment groups (6h and 24h after the third injection), the levels of the majority of the 1-deoxydihydroceramides and the levels of 1-deoxysphingosine were significantly reduced compared to the levels in the control group (Suppl. Table 8 and Fig. 5). Moreover, the levels of L-serine-derived canonical sphingoid bases and their phosphate derivatives, dihydrosphinganine-1-phosphate and S1P, were significantly reduced (Suppl. Table 9). C14, C18, and C18:1 ceramides also showed reduced levels in the sciatic nerve samples of the docetaxel-treated mice compared to controls. These data show that in the PNS, the DRG, where the somas of the sensory neurons are located, and the sciatic nerve, which is comprised by myelinated and non-myelinated axons of motor and sensory neurons, the effects of docetaxel on the sphingolipid metabolism is not uniform.

Figure 5. 1-Deoxydihydrosphingolipid and 1-deoxySL species down-regulated in the sciatic nerve of mice treated with docetaxel.

Figure 5.

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Mice (C57BL/6OlaHsd) were intraperitoneally injected three times (with four-week intervals) with docetaxel (cumulative dose 45mg/kg). Sciatic nerves were harvested 6h (three mice) and 24h (four mice) after the third injection of docetaxel or from the control group (three mice). Lipids were extracted from the isolated nerves and subjected to targeted mass spectrometry analyses of 1-deoxySL. Each treatment group consisted of three or four mice. The statistical analyses are presented in Suppl. Table 8. A/ C18 1-deoxydihydroceramide; B/ C20 1-deoxydihydroceramide; C/ C22 1-deoxydihydroceramide; D/ 1-deoxysphinganine; E/ 1-deoxysphingosine. The box plots were generated in Sigma plot 14.0 (Systat Software in San Jose, CA). For the treatment groups of three samples each line represents the actual lipid measurement. For the treatment group with four samples, the whiskers represent the higher and the lower value of the lipid measurement.

S1P rescues 1-deoxysphingosine neurotoxicity in primary DRG neurons

In an earlier study using Vero cells, it was shown that 1-deoxysphinganine (spisulosine) treatment resulted in changes in cell shape due to actin stress fiber disassembly (Cuadros et al. 2000). Notably, actin dynamics are involved in neurite elongation and retraction (Chen et al. 2012). We tested whether treatment with 1-deoxysphingosine (500nM for 6h) would affect the actin label and the neurite morphology in primary DRG neurons. Incubation of DRG neurons with 1-deoxysphingosine prevented neurite outgrowth when compared to vehicle controls (Fig. 6A–F) and led to actin-labeled neurite swellings (arrows in Fig. 6F, enlarged in 6J, and quantified in 6K). In the above mentioned study in Vero cells (Cuadros et al. 2000), pretreatment with lysophosphatidic acid (LPA) prevented actin stress fiber disassembly caused by 1-deoxysphinganine (spisulosine) treatment. LPA, as well as S1P, are second messengers and ligands to specific GPCR (reviewed in (Choi & Chun 2013)). Cytoskeleton regulation has been shown downstream of both LPA and S1P signaling. Thus, we next tested whether adding S1P (500nM) would attenuate the neurotoxic response to 1-deoxysphingosine in primary DRG neurons. Our results showed that adding S1P (500nM) to the 1-deoxysphingosine treatment restored neurite outgrowth (Fig. 6G–I) and significantly reduced neurite swelling (quantified in 6K). Our results suggest that S1P signaling can rescue 1-deoxysphingosine neurotoxic effect on DRG neurites. Time-lapse imaging revealed that the labeled actin in the control and in the combined treatment of S1P and 1-deoxysphingosine demonstrated movement toward the tips of the neurites over time (Suppl. movie 1 and 2, respectively). In contrast, the 1-deoxysphingosine treatment alone resulted in movement that was towards the soma (Suppl. movie 3). These results suggest that 1-deoxysphingosine might lead to impaired actin transport in the DRG neurites, which is corrected by S1P co-treatment.

Figure 6. Sphingosine-1-Phosphate (S1P) rescues 1-deoxysphingosine neurite swelling in primary DRG neurons.

Figure 6.

Figure 6.

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The DRG neurons were isolated from C57BL/6 mice and cultured for 48h before labeled with SirActin (Cytoskeleton) for 6h and subsequently treated with Huzzah® conjugated lipids (Avanti Polar Lipids) for an additional 6h. Time-lapse imaging was perform on Nikon epifluorescence microscope. A – C/ Huzzah® control; D – F/ Huzzah®1-deoxysphingosine (500nM); white arrows in F show the neurite swellings; G – I/ Huzzah®1-deoxysphingosine (500nM) + Huzzah® S1P (500nM); J/ enlarged image from F; K/ neurite swellings quantification was performed blindly with Nikon Elements analysis software. The results are from three independent experiments; i.e. separate DRG neurons isolations. In each experiment, three individual neurons were imaged for each treatment and at least three neurite regions of interest were selected for each neuron. Statistical analyses; control vs. 1-deoxysphingosine (500nM) p<0.001; 1-deoxysphingosine vs. 1-deoxysphingosine (500nM) + S1P (500nM) p<0.001 (ANOVA, Sigma plot).

Discussion

Our current work follows our previous study in breast cancer patients treated with a taxane (paclitaxel), where we reported an association of specific very-long chain plasma 1-deoxySL (with C22:1 and C24 fatty acid moiety) with patients’ peripheral neuropathy symptoms (Kramer et al. 2015). The data from our new study in the DRG of mice treated with a taxane (docetaxel), also resulted in the disruption of sphingolipid metabolism, leading to the upregulation of very-long chain 1-deoxySL, i.e. C22 and C24 (Suppl. Tables 1 and 2, and Fig. 3). In addition, our present results corroborated our previous finding in cells that taxane-induced upregulation of 1-deoxySL is due to the upregulation of SPT (Fig. 4) (Kramer et al. 2015). Taken together, the results from our two studies suggest that taxanes lead to the upregulation of SPT and of levels of specific very-long chain 1-deoxySL. In the current study, we also measured the levels of the canonical L-serine-derived dihydroceramides and ceramides. Interestingly, their taxane-induced upregulation was not individual species selective (Fig. 1; Suppl. Tables 4 and 5). In mammals, the fatty acid chain length of ceramides is determined by six ceramide synthases (CerS1–6) (reviewed in (Cingolani et al. 2016). The production of C22 and C24 ceramide species is catalyzed by CerS2 or CerS4. Considering that in the same DRG sample, taxane-induced upregulation of L-serine-derived ceramides was not selective; the upregulation of particular very long chain 1-deoxySL is not likely due to the upregulation of CerS2 and CerS4. It remains to be investigated in future studies if the selective upregulation of very-long chain 1-deoxySL is due to differential affinity of the individual CerS isoforms to 1-deoxysphinganine and 1-deoxysphingosine substrates. If true, this would result in the channeling of 1-deoxysphingoid bases into particular, very-long chain 1-deoxydihydroceramide and 1-deoxyceramide species.

It is possible that the increased levels of L-serine-derived ceramide in the DRG of docetaxel-treated mice results in increased levels of complex sphingolipids, such as sphingomyelin and gangliosides. Investigating ganglioside levels in future studies will be of a particular interest, considering that antiganglioside antibodies (e.g. anti-GM1, anti-GD1a, or anti-GD1b) were implicated in autoimmune peripheral neuropathies (recently reviewed in (Goodfellow & Willison 2018)). Moreover, the use of anti-GD2 antibody for a cancer immunotherapy has been shown to cause peripheral neuropathy (recently reviewed in (Patel & Spassieva 2018). The focus of our current study is the effect of docetaxel on 1-deoxySL levels, which have been shown by others and by us to be neurotoxic (Guntert et al. 2016, Penno et al. 2010, Spassieva et al. 2016). Nevertheless, we do not exclude that taxanes, by upregulating SPT and ceramide levels, affect ganglioside metabolism as well, which could potentially also contribute to neurotoxicity.

A somewhat unexpected result was that in the sciatic nerve, docetaxel treatment led to decreased levels of both L-alanine-derived 1-deoxySL and L-serine-derived ceramides (Suppl. Tables 8 and 9; Fig. 5). This is the opposite of our results from DRG (discussed above) where we observed increased levels due to docetaxel treatment. This difference can be due to the different permeability of taxanes into the DRG vs. the sciatic nerve. Several studies have shown that DRG accumulate paclitaxel because they are more easily accessible to outside agents than the sciatic nerve or the spinal cord (Cavaletti et al. 1997, Cavaletti et al. 2000, Abram et al. 2006). The blood-nerve barrier of the sciatic nerve is likely a sufficient obstacle for taxanes, similar to the blood-brain barrier in the CNS (Reinhold & Rittner 2017). Therefore, it is likely that taxanes directly affect sphingolipid metabolism in the DRG, leading to SPT and 1-deoxySL upregulation. The effects on the sphingolipid metabolism in the axons in the sciatic nerve, on the other hand, are likely indirect.

According to current clinical data, the major pathobiology of taxane-induced neuropathy is dying of the distal axonal endings of peripheral neurons (Argyriou et al. 2008, Fukuda et al. 2017, Landowski et al. 2016). The terminals of the sensory nerves are not protected by the blood-nerve barrier as the rest of the axons and are likely affected directly by toxic agents, such as taxanes. It remains to be investigated whether taxanes can directly affect 1-deoxySL levels in the c-fiber nerve endings, which will depend on future advances of the in situ methods for measuring them. However, as our previous study showed, systemic taxane administration led to increased plasma levels of certain 1-deoxySL that associated with peripheral neuropathy symptoms (Kramer et al. 2015). Therefore, it is possible that 1-deoxySL produced elsewhere in the body are transported through the plasma to affect unprotected c-fiber nerve endings.

Our in vitro results with primary DRG neurons show that exogenous administration of 1-deoxysphingosine could lead to neurite swellings and retraction, which was prevented by co-treatment with S1P (Fig. 6). Interestingly, S1P levels did not change because of docetaxel treatment, raising the question whether the balance between the levels of 1-deoxySL and S1P is important for neuronal homeostasis. Considering the interconvertibility of the sphingolipid pathway metabolites it remains to be tested in future studies whether exogenous 1-deoxysphingosine itself is neurotoxic or it would first require an uptake and a conversion to 1-deoxyceramides as suggested in a previous study (Alecu et al. 2017b). S1P can also be potentially up taken and converted to other bioactive sphingolipid metabolites such as ceramide-1-phosphate, although as reviewed recently direct acylation of S1P to ceramide-1-phospahte has not been shown and therefore unlikely (Presa et al. 2020). As discussed above (see introduction), S1P is a ligand to five S1PRs, which convey its cellular signaling effects. Considering that S1P signaling has been shown to affect the cytoskeleton (Gandy et al. 2013), a recent work suggesting 1-deoxySL are implicated in cytoskeletal regulation (Guntert et al. 2016), and our current data (Fig. 6 and suppl. Movies 1–3), it is likely that both, S1P and 1-deoxySL play roles in regulation of cytoskeleton and actin cytoskeleton in particular. Therefore, when the levels of those lipids are altered due to disruptions of sphingolipid homeostasis, as in the case of taxane treatment, this can over time lead to morphological changes at the distal axonal endings and ultimately neuropathy. Importantly, our data suggest that exploring sphingosine-1-phosphate signaling can identify targets to address taxane-induced neuropathy.

Supplementary Material

Table S1-S9
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movie S2
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movie S3
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Acknowledgments

We would like to thank the Department of Physiology (Chair Dr. Alan Daugherty) at the University of Kentucky (Lexington, KY, USA) for the support of this work and the Lipidomics core facility at the Medical University of South Carolina (Charleston, SC, USA) for the mass spectrometry measurements of sphingolipids. This work was in part supported by NIH grants R01NS62306 to BKT, R01AG034389 to EB, R56AG064234 to EB, and Department of Veteran Affairs grant I01 BX003643 to EB.

Abbreviations:

CerS

ceramide synthase

1-deoxySL

1-deoxysphingolipids

DRG

dorsal root ganglia

LC/MS/MS

liquid chromatography tandem mass spectrometry analyses

LPA

lysophosphatidic acid

PFA

paraformaldehyde

PNS

peripheral nervous system

PBS

phosphate buffered saline

RRID

Research Resource Identifier

SPT

serine palmitoyltransferase

S1P

sphingosine-1-phosphate

S1PR

sphingosine-1-phosphate receptor

Footnotes

Conflict of interest disclosure. The authors declare no conflicts of interest.

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Associated Data

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Supplementary Materials

Table S1-S9
NIHMS1571417-supplement-Table_S1-S9.pdf (149.1KB, pdf)
movie S1
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movie S2
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movie S3
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