Brain Advance Access originally published online on February 4, 2004
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Brain, Vol. 127, No. 3, 671-679, 2004
© 2004 Guarantors of Brain
doi: 10.1093/brain/awh078
Calpain inhibition protects against Taxol-induced sensory neuropathy
Departments of 1 Neurology and 2 Pathology, Emory University School of Medicine, Atlanta, GA, and 3 School of Chemistry and Biochemistry, Georgia Institute of Technology, Atlanta, GA, USA
Correspondence to: Jonathan D. Glass, MD, Emory Center for Neurodegenerative Disease, Whitehead Biomedical Research Building, 615 Michael Street, 5th Floor, Mailstop 194100700, Atlanta, GA 30322, USA E-mail: jglas03{at}emory.edu
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Taxol is a highly effective anticancer agent that causes peripheral neuropathy as its major toxic side effect. The neuropathy is characterized by degeneration of sensory axons that may be severe enough to be dose limiting. Axonal degeneration involves the activation of the calcium-activated proteases calpains, and here we tested whether systemic inhibition of calpains with the peptide
-ketoamide calpain inhibitor AK295 can reduce the clinical and pathological effects of Taxol in a rodent model of Taxol neuropathy. In mice with Taxol neuropathy, AK295 reduced the degree of axonal degeneration in sensory nerve roots, and improved clinical measures of neuropathy, including behavioural and electrophysiological function. These findings were consistent for both 3- and 6-week models of neuropathy. In vitro, Taxol caused activation of both calpains and caspases in PC12 cells. AK295 inhibited the activation of calpains but did not interfere with the antimitotic effects of Taxol on microtubules, nor did it inhibit caspase-mediated cell death. These data implicate calpains in the pathogenesis of Taxol neuropathy, and demonstrate that AK295 can prevent axonal degeneration and clinical neuropathy in mice. In addition, AK295 did not interfere with the primary antineoplastic effects of Taxol on microtubules and cell death, suggesting that systemic calpain inhibition may be a good strategy for preventing neuropathy in patients being treated with Taxol.
Key Words: axon; axonal degeneration; calpain; paclitaxel; peripheral neuropathy
Abbreviations: DMEM = Dulbecco’s modified Eagle’s medium; DMSO = dimethyl sulfoxide; DRG = dorsal root ganglia
Received August 21, 2003. Revised November 12, 2003. Accepted November 13, 2003.
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Introduction |
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Peripheral neuropathy is a major dose-limiting complication of commonly used anticancer agents, including vincristine, cisplatin and paclitaxel (Taxol). Taxol, a microtubule toxin derived from the western yew tree, is particularly effective against solid tumours, but causes a predominantly sensory neuropathy that can be severe enough to necessitate cessation of treatment (Black, 1987
; Kaplan et al., 1993; Rowinsky et al., 1993; Sahenk et al., 1994). The neuropathy is characterized by degeneration of sensory axons, manifesting clinically as numbness, pain and loss of balance. Taxol causes a similar sensory neuropathy in rodents, which provides a useful experimental model for the treatment of peripheral neuropathies (Cavaletti et al., 1995; Authier et al., 2000).
Calpains are ubiquitous cytosolic proteolytic enzymes involved in both physiological and pathological cellular functions (Croall and DeMartino, 1991). They are calcium-dependent enzymes belonging to the family of cysteine proteases. Limited activation of calpains results in modification or activation of protein receptors, enzymes and cytoskeletal proteins (Nixon, 1989; Melloni et al., 1992). Pathological cellular insults lead to more generalized calpain activation, resulting in cytoskeletal degradation and cell death. In the nervous system, calpain activation is believed to be responsible for the calcium-mediated cell injury seen in ischaemic stroke (Bartus et al., 1995), spinal cord injury (Li et al., 1995), closed head injury (Saatman et al., 1996a) and Wallerian degeneration (Kamakura et al., 1983).
Calpain inhibition protects against neuronal loss and improves neurological function in several models of nervous system injury (Bartus, 1997). Administration of calpain inhibitors prior to injury improves pathological and behavioural outcomes in animals subjected to head trauma (Saatman et al., 1996b), spinal cord contusion (James et al., 1998) and focal brain ischaemia (Bartus et al., 1994). In an in vitro dorsal root ganglion (DRG) model, we showed that the reversible peptide -ketoamide calpain inhibitor AK295 prevents axonal degeneration caused by axotomy or exposure to vincristine (Wang et al., 2000). Here we extend our findings to the clinically relevant, whole-animal model of Taxol neuropathy, demonstrating that systemic administration of AK295 significantly prevents the behavioural, electrophysiological and pathological effects of Taxol.
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Material
Cell culture materials were from Gibco/Invitrogen (www.lifetech.com). Taxol, Cremophor/EL and antibodies to
-tubulin were from Sigma (www.sigmaaldrich.com). Calpain substrate was from Bachem (www.bachem.com). Animals were purchased from Charles River Laboratories (www.criver.com). The Emory University Institutional Animal Care and Use Committee approved all animal protocols.
Taxol-induced axonal degeneration in rat DRG culture
DRG from E15 rats (Charles River) were cultured as described previously (Wang et al., 2000). Briefly, ganglia were dissected and stripped from connective tissue into L15 medium (Gibco), washed twice with phosphate-buffered saline (PBS) and plated in collagen-coated dishes containing Dulbecco’s modified Eagle’s medium (DMEM) with 1% N2 supplement and 7S nerve growth factor (100 ng/ml). Cultures were kept at 37°C, 5% CO2. After 5 days of growth, media was changed to that containing test agents (Taxol, AK295), and DRG remained in culture for an additional 10 days. Taxol was dissolved in Cremophor EL/ethanol (50:50); final concentration of Cremophor EL and ethanol in cultures was <0.0001%. AK295 was dissolved in dimethyl sulfoxide (DMSO), with a final concentration of DMSO in culture of 0.05%. This amount of DMSO demonstrated no effects on DRG growth (data not shown).
Serial images of DRG were captured on day 0, 4, 8 and 10 and were analysed as described previously (Wang et al., 2000). The area of the DRG halo at each time point was normalized to the area measured on day 0 (day test agents added), enabling each DRG to serve as its own control. Data were subjected to ANOVA, with post-test correction for multiple comparisons.
Taxol neuropathy and AK295 treatment in mice
Eight-week-old female C57BL/6J mice (Jackson Laboratories, Bar Harbor, MN, USA) were separated into four treatment groups as outlined in Fig. 1. All groups were treated with Taxol, and two groups were treated with a combination of Taxol and AK295. Three- and 6-week protocols were investigated. Taxol was dissolved 50:50 in Cremophor EL/ethanol and diluted 1:1 with saline; final concentration was 7.5 mg/ml. As described previously (Wang et al., 2002), each Taxol treatment consisted of three injections of 60 mg/kg into the jugular vein on an every other day schedule. This protocol was based on that provided by Mimura et al. (2000) in the original description of Taxol neuropathy in mice. For each injection the jugular vein was visualized and 50 µl of Taxol/Cremophor was injected over 3–5 min using a syringe pump, after which the skin was closed with a surgical clip. The 3-week groups received one Taxol treatment and the 6-week groups received two Taxol treatments. Control groups were treated with the Cremophor diluent only.
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AK295 [Z-leu-Abu-(CH2)3-4-morpholinyl] was synthesized as described previously (Li et al., 1996b
). The AK295 treatment groups received subcutaneous injections of AK295 (48 mg/kg) with each Taxol injection. After the last Taxol injection, a 100 µl Alzet pump (Alza Corporation, Mountain View, CA, USA) filled with AK295 (84 mg/ml in DMSO/polyethylene glycol, PEG 300, 1:1) was surgically implanted under the back skin. These pumps are designed to deliver drug at a rate of 6 µl/day for 14 days, translating into 0.504 mg/day, or 24 mg/kg/day for an average 18 g mouse. For the 6-week groups, the protocol for Taxol and AK295 treatment was repeated (subcutaneous AK295 with each Taxol injection) and a new pump was implanted for the remainder of the study. Control animals received initial injections with diluent only, and received pumps containing diluent only. Animals were killed by perfusion with 4% paraformaldehyde (in 0.1 M PBS buffer, pH 7.4) at the time points depicted in Fig. 1. The nerve roots (L4 dorsal and ventral) were harvested and post-fixed overnight in 5% buffered glutaraldehyde at 4°C. Nerve roots were rinsed with PBS buffer, processed by standard methods, and embedded in plastic for light microscopy. Sections of 780 nm were stained with toluidine blue for microscopy study and image analysis.
Image analysis
Images of dorsal and ventral roots (125x) were captured using a Kodak DCS-5 digital camera attached to an Olympus BH-2 microscope. Multiple overlapping images were captured, including all axons within the cross section. These images were combined into a montage so that the individual nerve fibres did not appear more than once. Images were analysed using ImagePro software (Media Cybernetics, Silver Spring, MD, USA) running on a Gateway personal computer. All myelinated axons were counted. Axonal density was calculated by dividing the number of axons by the area of nerve cross-section. The diameter and area of each remaining axon was measured by tracing the inner border of myelin. All data were subjected to ANOVA (analysis of variance).
Behavioural testing
To evaluate changes in neuromuscular function, animals were subjected to testing on a Rotarod apparatus (Columbus Instruments, Columbus, OH, USA) before Taxol treatments and prior to killing. The initial speed was set at 1.6 rpm with acceleration rate of 4 rpm/min. Animals were acclimated to the Rotarod for three consecutive days before the test date. The test was repeated three times during each testing session with at least 2 min of rest between each test. The best performance of each session was recorded. Percent changes were calculated and analysed using ANOVA with post-test comparisons.
Electrophysiology
As previously described (Wang et al., 2002), nerve conduction studies were performed using standard equipment (Nicolet, Madison, WI, USA) on anaesthetized animals on the same schedule as Rotarod testing. For hind limb recording, the recording electrodes were inserted into the interosseous muscles of the left foot; stimuli were administered at the ankle and at the hip (sciatic notch) close to the tibial and sciatic nerve, respectively. A ground electrode was inserted subcutaneously into the tail. Compound muscle action potentials were recorded (maximum) and nerve conduction velocities were calculated. For tail nerve recording, the recording electrodes were placed at the base of the tail, keeping the anode and the cathode 
5 mm apart. Stimuli were administered 4–5 cm distal. A ground electrode was placed in between the stimulus and recording electrodes. The sensory nerve action potential was averaged over 50–80 stimuli, and the amplitude was recorded. Sensory conduction velocity was also calculated.
Calpain activation assay
Calpain activity was measured in PC12 cells using the methods described by Bronk and Gores (1993) and modified by Weber et al. (2002). PC12 cells were grown for 24 h in DMEM with 10% horse and 5% fetal calf serums. Calpain activity in response to Taxol was assessed in relation to dose (1, 10, 50 or 100 ng/ml for 24 h) and of time of exposure (10 ng/ml of Taxol for up to 48 h). After exposure, cells were suspended in KRH buffer (25 mM Na-HEPES, 115 mM NaCl, 5 mM KCl, 1 mM KH2PO4, 1.2 mM MgSO4, 2 mM CaCl2, 0.2% bovine serum albumin, pH 7.4) and 2 ml of cell suspension was transferred to a test tube. The reaction was started by adding 100 µl of the cell-permeable calpain substrate (Suc-Leu-Leu-Val-Tyr-AMC; 0.84 mg/ml). The suspension was mixed immediately and incubated at 37°C for 15 min. The reaction was stopped by adding 100 µl of 0.4 N HCl. After sonication and centrifugation, free AMC in the supernatant was measured using a microplate reader (
ex 355/em 460). A standard curve was constructed using AMC (7-amino-4-methylcoumarin standard. Calpain activity was expressed as AMC concentration (pM) per 106 cells.
Imaging of microtubule aggregates
The method was adapted from that described by Giannakakou et al. (1998). PC12 cells were cultured on collagen-coated coverslips for 48 h and then treated for 24 h with Taxol or AK295. After treatment cells were fixed with 4% paraformaldehyde for 30 min and post-fixed with pre-cooled methanol for 5 min at –20°C. The cells were then rinsed with PBS and stained with antibody to -tubulin using standard immunofluorescence staining methods. The cells were imaged on a Zeiss LSM 510 confocal microscope for identification of microtubule aggregates and mitotic figures.
Cytotoxicity assay
PC12 cells were cultured in 96-well plates containing DMEM supplemented with 10% horse serum and 5% fetal calf serum, 200 µl/well. At 80% confluence, 100 µl medium was replaced with 50 µl Sytox® (Molecular Probes, www.molecularprobes.com; final concentration 4 µM) and 50 µl of the experimental compounds (Taxol 10, 100, 200 or 1000 ng/ml, AK295, 50 µM). The caspase inhibitor JG36 (Cbz-Asp-Glu-Val-AAsp-EP-COOEt) was synthesized in our laboratory (by J.C.P.). JG36 is highly active against caspase-3 and has little activity against other cysteine proteases (Asgian et al., 2002).
Fluorescence (ex 485/em 538) was measured immediately and at scheduled time points for up to 24 h. After the last reading, 50 µl of 4% paraformaldehyde was added to each well and the plate was kept at 4°C for 1 h followed by a final reading for the maximal amount of fluorescence release. The experimental protocol was repeated three times.
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Results |
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Taxol-induced axon degeneration in DRG culture
Exposure to Taxol caused dose-dependent axonal degeneration in cultured dorsal root ganglia. Doses of 25 ng/ml or greater caused rapid axonal degeneration; a dose of 5 ng/ml did not cause obvious degeneration, but did slow axonal growth (Fig. 2). Degeneration occurred in a distal to proximal pattern (‘dying back’) similar to that seen in DRG cultures exposed to vincristine (Wang et al., 2000
). Addition of AK295 (50 µM) to the culture media provided 50% protection against axonal degeneration induced by 25 ng/ml Taxol for up to 8 days, as measured by the area of the DRG halo (data not shown).
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Taxol-induced axonal degeneration and peripheral neuropathy in mice
Taxol caused dose-dependent axonal degeneration in mice. Two doses (3 x 30 mg/kg and 3 x 60 mg/kg) were tested. The low dose caused axonal degeneration in relatively few fibres, with inconsistent numbers of degenerating fibres (data not shown). The higher dose caused degeneration of a significant number of sensory fibres (Fig. 3), which was reproducible or suitable for our purpose of quantitative analysis. Using this paradigm, Taxol did not cause axonal degeneration in motor fibres.
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Nineteen mice were used for the 3-week protocol: control (four), Taxol (seven) and Taxol plus AK295 (eight). Ten mice were used for the 6-week protocol: control (four), Taxol (three) and Taxol plus AK295 (three). The animals treated with Taxol or Taxol plus AK295 showed weight loss of 1–3 g in the first week, but regained a normal growth rate and were in good health for the remainder of the study (Fig. 4). Rotarod and electrophysiological measures supported the presence of peripheral neuropathy (Fig. 4), and pathological analysis of dorsal roots demonstrated significant loss of myelinated fibres at the 3-week time point (Table 1). There was little evidence of further progression of neuropathy in animals receiving a second Taxol treatment during the fourth week and evaluated at the 6-week time point. Pilot experiments, however, demonstrated almost full recovery of myelinated fibre numbers at 6 weeks in animals not receiving a second Taxol treatment (data not shown), suggesting that the second Taxol treatment had the effect of maintaining the neuropathy in animals that would have otherwise recovered. Analysis of fibres grouped by diameter demonstrated that axonal loss was most prominent in larger fibres (Fig. 5), as previously demonstrated in Taxol neuropathy in humans and rats.
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AK295 protects against Taxol neuropathy
AK295 treatment was protective against Taxol neuropathy by all measures. Behavioural and electrophysiological testing showed protection in the AK295 group at 3 weeks that persisted to the 6-week time point (Fig. 4). Comparing pre- and post-treatment measurements, animals treated with diluent only improved by
20% on the Rotarod (not shown), whereas performance in Taxol-treated mice was reduced to 60% of baseline. Taxol + AK295-treated mice remained at baseline levels. Similar results were obtained from sensory nerve conduction studies. Tail sensory nerve action potential in Taxol-treated mice was reduced to 50% of baseline, whereas Taxol + AK295-treated mice showed no reduction in sensory amplitudes. There were no significant effects of Taxol on motor conduction studies in either sciatic or tail nerves (not shown). Pathologically, the degree of axonal degeneration was less in AK295-treated mice compared with mice treated with Taxol only (Fig. 3). Quantitative analysis demonstrated an increase in fibre number and density at both 3 and 6 weeks in the AK295 group (Table 1). Mean fibre diameter was also increased toward normal in these groups (Table 1). Subgroup analysis of the effects of Taxol and AK295 by fibre size demonstrated the large-fibre predominance of Taxol toxicity and the relative protection by AK295 in these larger fibres (Fig. 5).
Taxol-induced calpain activation in PC12 cells
PC12 cells were used to demonstrate that exposure to Taxol can induce calpain activation and that AK295 inhibits calpain activity. In PC12 cells there was both time- and dose-dependent increase in calpain activity as measured by cleavage of a synthetic calpain substrate (Fig. 6). AK295 inhibited calpain-mediated cleavage of the substrate. It is of interest to note that AK295 reduced the baseline calpain activity measured in non-treated cells as well, without causing any apparent toxicity.
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Taxol-induced tubulin aggregation and cell death
The antineoplastic effects of Taxol are based on its capacity to bind and stabilize microtubules, leading to mitotic arrest, activation of caspases and cell death (Blagosklonny et al., 1996
). We were concerned that inhibition of calpains with AK295 might interfere with Taxol-mediated cell death. To address this issue we used PC12 cells to assess the effect of AK295 on the formation of tubulin bundles and caspase-mediated cell death in response to Taxol exposure. PC12 cells showed aggregation of -tubulin after exposure to Taxol with or without addition of AK295 (Fig. 7). The frequency of mitotic arrest was also unchanged in cells treated with AK295. The Sytox® cytotoxicity assay showed cell death after exposure to Taxol that was unaffected by the presence of AK295 (Fig. 8). Addition of a caspase-3 inhibitor reduced cell death to control levels.
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Discussion |
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The findings in this study demonstrate that AK295 is protective against Taxol-induced axonal degeneration and peripheral neuropathy in a clinically relevant whole-animal model. AK295 treatment significantly improved the outcome of Taxol neuropathy as measured behaviourally, electrophysiologically and pathologically. The effect was apparent in both short- and long-term experimental protocols, suggesting that AK295 does not merely delay neuropathy, but also is protective against both axonal degeneration and neuropathy.
Taxol neuropathy in humans is a symmetrical, length-dependent, sensory predominant axonal neuropathy (Lipton et al., 1989). The occurrence of Taxol neuropathy can be predicted based on the cumulative dose, although the neuropathy may appear earlier and at lower doses in patients with pre-existing neuropathies (Windebank, 1999). This complication is frequently the dose-limiting toxicity of chemotherapy when higher doses of Taxol are administered. This animal model faithfully reproduces the human condition, and the finding of protection with systemic calpain inhibition warrants some optimism regarding similar approaches in humans allowing for sustained treatment with higher doses of this effective chemotherapeutic agent.
Current theories regarding the pathogenesis of Taxol neuropathy are not intuitively consistent with our findings of protection by calpain inhibition. The neurotoxicity of Taxol is presumed to result from its ability to impair axonal transport through stabilization and excessive polymerization of microtubules (Roytta and Raine, 1985; Nakata and Yorifuji, 1999; Theiss and Meller, 2000), a mechanism that is central to hypotheses of toxin-induced peripheral neuropathies (Griffin and Watson, 1988; Windebank, 1999). Our data, however, demonstrate that Taxol can also activate calpains. Calpain activity in PC12 cells increased in a time- and dose-dependent fashion in response to Taxol, and AK295 inhibited this Taxol-induced calpain activation. Given these findings, along with our demonstration that AK295 prevented Taxol neuropathy in mice, we conclude that the mechanism of Taxol neuropathy involves the activation of calpains, either as a primary or a secondary phenomenon.
Calpain activation in axons is a well-recognized feature of axonal degeneration and probably plays a significant role in the pathogenesis of some neuropathies (Schlaepfer and Zimmerman, 1984; Wang et al., 2000). There are no data linking the disruption of axonal transport with the activation of calpains, but the current findings along with our previous data on experimental vincristine neuropathy (Wang et al., 2000) suggest a relationship between the two. It may be that calpain activation is a downstream effect of many cellular injuries, including disrupted axonal transport that leads to cytoskeletal degradation and cell death. Thus, calpain inhibition may be protective in many disorders where axonal degeneration is a prominent feature.
A concern for the use of AK295 for prevention of Taxol neuropathy is the potential for interference with the primary antineoplastic effects of Taxol. We addressed this issue in two ways. First, staining of Taxol-treated PC12 cells with tubulin antibodies showed no difference in the morphological footprints of microtubule bundling and mitotic arrest with the addition of AK295. Secondly, the cytotoxicity assay showed no effect of AK295 on cell killing by Taxol, but a protective effect of our experimental caspase inhibitor. These results suggest that Taxol has differential effects on dividing cells and on axons: the antimitotic/anticancer effect is mediated by the activation of caspases (Oyaizu et al., 1999), whereas axonal toxicity appears to be mediated by activation of calpains.
Calpains have been shown to be involved in several acute and chronic neurological disorders including focal brain ischaemia (Siman et al., 1996), head and spinal cord trauma (Li et al., 1996a; Saatman et al., 1996a), Parkinson’s and Alzheimer’s disease (Nixon et al., 1994; Mouatt-Prigent et al., 1996; Ray et al., 2000), and peripheral neuropathies (Wang et al., 2000). Calpain activation probably occurs due to the sustained elevation of intracellular calcium that is a common feature of models of neuronal injury (Nixon, 1989; Meldrum and Garthwaite, 1990; Caner et al., 1993; Choi, 1993; LoPachin and Lehning, 1997; for a review see Bartus, 1997). The use of AK295 for treatment of experimental nervous system injury has thus far been restricted to acute models (Bartus et al., 1994; Saatman et al., 1996b). AK295 is a potent transition-state reversible inhibitor for both calpain I (KI = 0.14 µM) and calpain II (KI = 0.041 µM), and is a less effective inhibitor of other cysteine proteases such as cathepsin B (Li et al., 1996b). Here we show that chronic calpain inhibition with this agent is effective in a model of chronic axonal degeneration after exposure to a neurotoxic agent. We identified no toxic side effects of AK295 either in culture or in the animal, further encouraging the potential usefulness of this agent for long-term treatments.
Our findings of AK295 protection in Taxol neuropathy may have implications for a number of other disorders that are characterized by axonal degeneration over a chronic time course. We have already shown that this agent is effective against vincristine neuropathy in DRG culture, and suspect that chronic use of AK295 may protect against axonal degeneration in other peripheral neuropathies. Studies are underway to test AK295 for its promise in treating neuropathies and other neurodegenerative diseases.
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Acknowledgements |
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Supported by P01 NS40405 from the National Institutes of Health.
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