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Differential expression of ATP7A, ATP7B and CTR1 in adult rat dorsal root ganglion tissue
Molecular Painvolume 6, Article number: 53 (2010)
ATP7A, ATP7B and CTR1 are metal transporting proteins that control the cellular disposition of copper and platinum drugs, but their expression in dorsal root ganglion (DRG) tissue and their role in platinum-induced neurotoxicity are unknown. To investigate the DRG expression of ATP7A, ATP7B and CTR1, lumbar DRG and reference tissues were collected for real time quantitative PCR, RT-PCR, immunohistochemistry and Western blot analysis from healthy control adult rats or from animals treated with intraperitoneal oxaliplatin (1.85 mg/kg) or drug vehicle twice weekly for 8 weeks.
In DRG tissue from healthy control animals, ATP7A mRNA was clearly detectable at levels similar to those found in the brain and spinal cord, and intense ATP7A immunoreactivity was localised to the cytoplasm of cell bodies of smaller DRG neurons without staining of satellite cells, nerve fibres or co-localisation with phosphorylated heavy neurofilament subunit (pNF-H). High levels of CTR1 mRNA were detected in all tissues from healthy control animals, and strong CTR1 immunoreactivity was associated with plasma membranes and vesicular cytoplasmic structures of the cell bodies of larger-sized DRG neurons without co-localization with ATP7A. DRG neurons with strong expression of ATP7A or CTR1 had distinct cell body size profiles with minimal overlap between them. Oxaliplatin treatment did not alter the size profile of strongly ATP7A-immunoreactive neurons but significantly reduced the size profile of strongly CTR1-immunoreactive neurons. ATP7B mRNA was barely detectable, and no specific immunoreactivity for ATP7B was found, in DRG tissue from healthy control animals.
In conclusion, adult rat DRG tissue exhibits a specific pattern of expression of copper transporters with distinct subsets of peripheral sensory neurons intensely expressing either ATP7A or CTR1, but not both or ATP7B. The neuron subtype-specific and largely non-overlapping distribution of ATP7A and CTR1 within rat DRG tissue may be required to support the potentially differing cuproenzyme requirements of distinct subsets of sensory neurons, and could influence the transport and neurotoxicity of oxaliplatin.
ATP7A, ATP7B and CTR1 are copper transporting proteins that have evolved along with other components of copper regulatory pathways for delivering copper to essential cuproenzymes without releasing highly cytotoxic free copper ions [1, 2]. The P-type ATPases, ATP7A and ATP7B, both transport copper out of cells or into the trans-Golgi network , whereas CTR1 is a plasma membrane protein that functions as a high-affinity cellular copper uptake transporter . ATP7A, ATP7B and CTR1 exhibit cell-type specific expression in the brain and other tissues [5, 6], reflecting their requirements for copper to support the functions of diverse cuproenzymes, such as dopamine-β-monooxygenase and peptidylglycine α-amidating monooxygenase that convert dopamine to norepinephrine and synthesize neuropeptides, respectively . Disturbance of copper transporters causes neurodegeneration. For example, mutation of ATP7A and ATP7B causes Menkes and Wilson disease, respectively, both of which have serious neurological sequelae including mental retardation, seizures, developmental delay and ataxia .
Little is currently known about the expression of copper transporters in the dorsal root ganglia (DRG) that contain the cell bodies of primary sensory neurons. These neurons may require copper transport as they strongly express cuproenzymes, such as cytochrome C oxidase , Cu/Zn superoxide dismutase  and peptidylglycine α-amidating monooxygenase , and are sensitive to copper deficiency [11, 12]. In other cell types, copper transporters have been shown to have a role in controlling the cellular accumulation and cytotoxicity of platinum drugs, with CTR1 mediating platinum uptake into cells [13–15], and ATP7A and ATP7B transporting platinum out of cells or into specific sub-cellular compartments [16–20]. Platinum-based drugs, such as cisplatin and oxaliplatin, accumulate in DRG tissue [21–26], damage sensory neurons [21, 22, 24–33], and induce peripheral sensory neuropathies that limit their use in clinical cancer chemotherapy . In the current study, we investigated the expression of ATP7A, ATP7B and CTR1 in DRG tissue from adult rats, either healthy control animals or those treated with oxaliplatin or its drug vehicle. Neuronal atrophy was used as the endpoint for measuring the neurotoxicity of oxaliplatin in DRG tissues, as in previous studies [26, 29, 35–37]. We aimed to determine patterns of expression and localization of ATP7A and ATP7B within DRG tissue, in an extension to our recent study of CTR1 , and to relate the expression of these copper transporters to the neurotoxicity of oxaliplatin.
Copper transporter gene expression in DRG and other tissues
The expression of copper transporter genes in rat DRG tissue was determined by RT-PCR and qPCR in comparison to reference tissues (brain, spinal cord, liver, kidney and intestine). The RT-PCR (Figure 1) and qPCR (Table 1) findings corresponded well with each other. In all of the tissues, CTR1 had the highest mRNA levels, followed by ATP7A and then ATP7B had the least, except in liver where ATP7B levels were higher than ATP7A.
In DRG tissue, ATP7B mRNA was barely detectable with only faint or no bands visible on RT-PCR gels but in liver tissue bands were more clearly visible (Figure 1). ATP7B mRNA transcripts in DRG tissue were detected by qPCR in only two of six animals compared to all six animals for liver (Table 1). In DRG tissue, ATP7A mRNA was more readily detectable than ATP7B, with clearly visible bands on RT-PCR gels (Figure 1) and qPCR-detectable transcripts in all six animals (Table 1). ATP7A mRNA levels in DRG were similar to brain and spinal cord levels but higher than those in the non-neuronal reference tissues.
High levels of CTR1 mRNA were found in DRG, as in other tissues. RT-PCR gels showed clearly visible bands for CTR1 in all tissues (Figure 1). CTR1 mRNA transcripts was detectable by qPCR in all tissues, and in all animals, at levels higher than ATP7A and ATP7B (Table 1).
Copper transporter protein expression in DRG tissue
ATP7A had a specific pattern of distribution within rat DRG tissue, with intense cytoplasmic staining of the cell bodies of smaller DRG neurons as revealed by immunohistochemistry. The specificity of anti-ATP7A primary antibody was confirmed by Western blotting showing a protein band with the size 170 kDa on ATP7A immunoblots of DRG tissue homogenates from rats aged 4, 12 and 20 weeks (Figure 2A). Negative controls that excluded the primary antibody lacked specific immunoreactivity (Figure 2B 2a, inset). ATP7A immunohistochemistry of DRG tissue visualized by ABC-peroxidase revealed that this copper efflux transporter was most strongly expressed within smaller-sized DRG neurons that showed intense immunoreactivity in a punctuate pattern localised to the cytoplasm of their neuronal cell bodies (Figure 2B,a, and 2b). Other DRG neurons showed lighter and more diffuse cytoplasmic immunostaining for ATP7A, with occasional granular staining of the plasma membrane. No ATP7A immunoreactivity was apparent in the satellite cells, nerve fibres, or other non-neuronal tissue elements of the rat DRG. Furthermore, fluorescent immunohistochemistry showed that ATP7A immunoreactivity was mainly associated with smaller DRG neurons that did not overlap with DAPI-stained non-neuronal cells or with the pNF-H-immunoreactive larger neurons and nerve fibres (Figure 2B,c).
No specific immunoreactivity for ATP7B was found in rat DRG tissue when compared with a negative control (data not shown), even though the primary antibody (NB100-360, Novus Biologicals) detects ATP7B in rat liver .
CTR1 immunohistochemistry of rat DRG tissue showed a pattern of immunostaining that differed from ATP7A. The specificity of the Novus antibody for CTR1 immunohistchemistry has been previously determined by preabsorption assay with immunizing peptide using a hCTR1 A2780 human ovarian carcinoma cell line . This antibody, however, is unsuitable for use in Western blot analysis. Strong CTR1 immunoreactivity was associated with the plasma membrane and cytoplasmic vesicular structures of larger-sized DRG neurons, whereas only light staining appeared in the remaining neurons (Figure 3A,B), as previously described . Double label fluorescent immunohistochemistry provided further evidence of CTR1 and ATP7A primary localisation to neuronal cell bodies, and their distinct patterns of immunoreactivity and non-overlapping distribution within rat DRG tissue (Figure 3C).
Morphometry of ATP7A-positive and CTR1-positive neurons in DRG tissue from control and oxaliplatin-treated rats
Morphometric analysis of control animal DRG tissue showed that ATP7A and CTR1 were expressed by different neuronal subpopulations with differing size profiles (Figure 4; Table 2). For this analysis, strongly ATP7A-expressing DRG neurons were defined as those having intense diffuse or punctuate cytoplasmic staining and/or plasma membrane immunoreactivity to ATP7A. Those negative for strong ATP7A expression had no or low-intensity diffuse or punctuate cytoplasmic staining without plasma membrane immunoreactivity. Strongly CTR1-expressing DRG neurons were defined as those having intense plasma membrane and/or punctuate cytoplasmic immunoreactivity to CTR1. Those negative for strong CTR1 expression had only diffuse cell body immunoreactivity without plasma membrane or punctuate cytoplasmic immunoreactivity. DRG neurons with strong expression of ATP7A accounted for 35.1 ± 2.9% of the overall population of DRG neurons in control animals, whereas those with strong expression of CTR1 accounted for 10.9 ± 1.8% (P < 0.001). About 64.2 ± 6.9% of the strongly ATP7A-expressing neurons had cell bodies measuring < 750 μm2, but only 2.0 ± 1.3% of strongly CTR1-expressing neurons were of this size (P < 0.001). About 58.2 ± 16.1% of strongly CTR1-expressing neurons had cell bodies measuring >1750 μm2, but only 6.7 ± 2.5% of strongly ATP7A-expressing neurons were of this size (P < 0.001). The mean cell body area of strongly ATP7A-expressing neurons (767.1 ± 87.6 μm2) was significantly smaller than that of the strongly CTR1-expressing neurons (1936 ± 278 μm2; P < 0.001).
Oxaliplatin treatment did not significantly change the size profile of strongly ATP7A-expressing neurons, immunoreactivity pattern of ATP7A or CTR1, or the staining frequencies of ATP7A-or CTR1-positive cells (Figure 4A,B). In contrast, oxaliplatin treatment of animals caused atrophy of strongly CTR1-expressing DRG neurons, which showed a clear left-ward shift in their size distribution profile, reduction in their mean cell body areas from 1936 ± 278 μm2 to 1461 ± 64 μm2 (P < 0.01), a decrease in the percentage of large neurons measuring greater than 1750 μm2 from 58.2 ± 16.1% to 28.5 ± 5.4% (P < 0.01), and an increase in the percentage of medium sized neurons measuring between 750 to 1750 μm2 from 39.9 ± 15.9% to 66.4 ± 5.5% (P < 0.01) (Table 2).
This is the first description of the expression of copper-transporting P-type ATPases in DRG tissue from rats or any other animal species. Adult rat DRG tissue exhibited a specific pattern of expression of copper transporters with distinct subsets of sensory neurons intensely expressing either ATP7A or CTR1, but not both or ATP7B. Copper transporter mRNA levels in DRG were highest for CTR1, followed by ATP7A and barely detectable for ATP7B. ATP7A protein was detected in DRG tissue homogenates by Western blotting. ATP7A and CTR1 were detectable in DRG tissue by immunohistochemistry and were localised to the cell bodies of sensory neurons with little or no immunostaining of nerve fibres, satellite cells or other tissue elements, and without specific immunoreactivity for ATP7B. Neuronal immunoreactivity for ATP7A did not co-localise with CTR1, pNF-H or DAPI-stained satellite cells in double-label fluorescent immunohistochemistry studies, and their cell body size-profiles determined by morphometric analysis differed markedly from that of CTR1-immunoreactive neurons. Morphometric analyses of immunohistochemically defined subpopulations of cells in tissue sections is inherently subjective as qualitative interpretation of cell positivity and arbitrary definitions of positive and negative cells are required. However, strongly ATP7A-immunoreactive neurons accounted for about one third of the overall total population of DRG neurons, and were characterised by their small cell bodies and intense punctuate cytoplasmic immunostaining. In contrast, strongly CTR1-immunoreactive neurons accounted for about one tenth of the overall population of DRG neurons and were characterised by their large cell bodies, and intense plasma membrane and vesicular cytoplasmic immunostaining, as we recently described . Together, these findings show that ATP7A and CTR1 have neuron subtype-specific and largely non-overlapping distribution in adult rat DRG tissue suggesting that these copper transporters have distinct roles in supporting the functions of primary sensory neurons.
The physiological significance of differential expression of copper transporters by DRG neurons is unclear and requires further study. However, ATP7A and CTR1 may be required by distinct sub-types of DRG neurons to deliver copper to specific cuproenzymes vital for the synthesis of neuropeptides and ATP. ATP7A, for example, delivers copper to peptidylglycine alpha-amidating monooxygenase in cell types other than DRG neurons [40–42], but this cuproenzyme activity is required by DRG neurons for the synthesis of substance P [10, 43, 44]. Like ATP7A, substance P is primarily expressed by small DRG neurons [26, 45, 46], and the size of substance P-expressing DRG neurons is not altered by oxaliplatin treatment . This suggests the existence of a subset of sensory neurons that co-express ATP7A, peptidyl alpha-amidating monooxygenase and substance P to support neuronal functions requiring neuropeptide synthesis. In other cell types, the level of expression of CTR1 corresponds closely with the activity of cytochrome C oxidase [4, 47, 48], which is a cuproenzyme involved in oxidative phosphorylation ATP synthesis. Like CTR1, cytochrome C oxidase is expressed intensely by large-sized DRG neurons , which may have reduced capacity for glycolysis compared to small DRG neurons , consistent with their strong need for CTR1 to meet their high demands for copper delivery to cytochrome C oxidase and ATP synthesis via oxidative phosphorylation. In this way, the neuronal subtype-specific and largely non-overlapping distribution of ATP7A and CTR1 in DRG tissue may relate to specific cuproenzyme requirements by distinct subsets of primary sensory neurons.
Platinum antitumour drugs, such as oxaliplatin, are known for causing peripheral neuropathy by undefined mechanisms that might involve platinum accumulation within the DRG leading to atrophy or loss of peripheral sensory neurons [21–33, 50–52]. Atrophy of DRG neurons would be expected to lead to altered sensory nerve conduction velocities that characterise oxaliplatin-induced peripheral neuropathy because DRG cell body size, axonal calibre and nerve conduction velocity are strongly correlated [53–55]. Furthermore, our previous work has suggested that oxaliplatin may induce atrophy of specific subpopulations of DRG neurons by causing the loss of phosphorylated neurofilament heavy subunit, which is a cyto-skeletal protein that determines the calibre of large myelinated DRG neurons and their axons . In other cell types, the cellular accumulation and cytotoxicity of platinum drugs is controlled, at least in part, by copper transporters, with CTR1 transporting platinum drugs into cells [13–15], and ATP7A and ATP7B transporting platinum drugs out of cells or into specific subcellular compartments [16–20]. In the current study, we showed that oxaliplatin treatment of adult rats caused atrophy of the CTR1-immunoreactive subpopulation of DRG neurons without changing the size profile of the ATP7A-immunoreactive subpopulation of DRG neurons. It is possible that ATP7A expressing DRG neurons are less sensitive to oxaliplatin neurotoxicity because the high levels of ATP7A facilitate the cellular efflux of oxaliplatin reducing its availability for reactions with DNA or other key neurotoxicity targets. In contrast, DRG neurons expressing high levels of CTR1 would be expected to take up more oxaliplatin leading to toxic effects in this neuronal subtype. Thus we suggest the neuronal subtype-specific and largely non-overlapping distribution of ATP7A and CTR1 within DRG tissue influence the neurotoxicity of oxaliplatin by controlling its cellular accumulation and sub-cellular distribution within primary sensory neurons. If this is so, then oxaliplatin treatment could be expected to alter the expression, distribution and sub-cellular localisation of ATP7A and CTR1 as in other cell types [15, 20, 39, 56, 57] but no evidence for such a change was found in DRG tissue in the current study. The role of CTR1 and ATP7A in oxaliplatin neurotoxicity remains hypothetical but could be tested further in studies comparing the accumulation, sub-cellular distribution and neurotoxicity of oxaliplatin in CTR1-and ATP7A-expressing neuronal cells in vitro making use of a CTR1 inhibitor to block the neuronal uptake and neurotoxicity of oxaliplatin.
In conclusion, adult rat DRG tissue exhibits a specific pattern of expression of copper transporters with distinct subsets of peripheral sensory neurons intensely expressing either ATP7A or CTR1, but not both or ATP7B. The neuron subtype-specific and largely non-overlapping distribution of ATP7A and CTR1 within rat DRG tissue may be required to support the differing cuproenzyme requirements of distinct subsets of sensory neurons, and could influence the transport and neurotoxicity of oxaliplatin.
Animals and drug treatment
Age-matched, 12-week-old female Wistar rats were housed in a self-contained unit maintained at 22 ± 2°C, and set to 12 h dark-light cycles with access to food and water ad libitum. Twelve healthy untreated animals were used for Cu transporter expression study by immunoblotting, immunohistochemistry and PCR respectively. In addition, for treatment study, two groups of animals received intraperitoneal injections of either oxaliplatin (Eloxatin; Sanofi-Aventis, Bridgewater, NJ, USA) at a dose of 1.85 mg/kg (n = 13) or dextrose (n = 12), as vehicle control, twice weekly for 8 weeks between 1300 and 1500 h. All animal procedures were approved by the institutional Animal Ethics Committee (AEC No. R591).
Western blot analysis
Following euthanasia of animals with intraperitoneal injection of pentobarbitone (90 mg/kg body weight, Chemstock Animal Health, Christchurch, New Zealand), lumbar DRG tissues were dissected and homogenized using a Dounce homogenizer (Glas-Col, Terre Haute, IN, USA) for 3 min in a lysis buffer containing 250 mM sucrose, 1 mM EDTA, 1 mM EGTA, 0.5% NP-40, 0.1% SDS, and a protease inhibitor mixture (Complete Mini Protease Inhibitor Cocktail tablets; Roche Diagnostics, Indianapolis, IN, USA). The homogenate was centrifuged for 15 min at 500 ×g at 4°C to remove nuclei and large particulate matter, and the protein concentration of the resulting supernatant was determined by a bicinchoninic acid (BCA) assay as previously reported . Protein samples (40 μg) were heated at 95°C for 30 min, resolved in 8% SDS-PAGE, and then transferred to a nitrocellulose membrane (Amersham Pharmacia, Tokyo, Japan) using a Transblot SD apparatus (Bio-Rad, Hercules, CA, USA). Following blocking with 5% milk/bovine serum albumin solution, ATP7A was detected by chemiluminescence using anti-ATP7A antibody (1:1000, no. ab13995: Abcam, Cambridge, UK), horseradish peroxidase (HRP)-conjugated anti-chicken antibody (Sigma-Aldrich, St. Louis, MO, USA), and the ECL Advance Detection reagent (Amersham Biosciences, Buckinghamshire, UK). Beta actin was probed to determine the equal loading using anti-beta actin antibody (Abcam) and a HRP-conjugated anti-rabbit IgG antibody (Amersham).
Animals designated for Atp7a, Atp7b and Ctr1 RT-PCR analysis were euthanized with pentobarbitone as above described. The lumbar DRG, brain, spinal cord, liver, kidney and small intestine tissues were collected and homogenized in PureZol reagent for total RNA isolation using an Aurum Total RNA Fatty and Fibrous Tissue Kit (Bio-Rad). After digestion with DNase I (1 unit/μg, Bio-Rad), total RNA of each sample (0.25 μg) was reverse-transcribed into cDNA using a SuperScript first strand synthesis kit (Invitrogen, Carlsbad, CA, USA) according to instructions, followed by digestion with Ribonuclease H (Invitrogen) to remove the RNA templates. cDNA was amplified by PCR in a reaction mixture containing dNTP, MgCl2, Platinum Taq DNA polymerase (Invitrogen) and custom primers, using a GeneAmp 9700 PCR System (Applied Biosystems, Foster City, CA, USA) at 52°C for 40 cycles. Forward and reverse primers for rat Atp7a were: 5'-tag acg gca tgc att gta aat c-3' and 5'-tgg att tta cac ctg gct tct t-3'(amplicon of 375 bp); for rat Atp7b were 5'-att cca gga ctg tcc gtt cta a-3' and 5'-cac ttg ctc ctc tct gag gat t-3'(amplicon of 396 bp); for rat Ctr1 were: 5'-ttg gct tta aga atg tgg acc t-3' and 5'-cat aag gat ggt tcc att tgg t-3'(amplicon of 206 bp); and for rat glyceraldehyde-3-phosphate dehydrogenase (GAPDH): 5'-tgc tga gta tgt cgt gga gtc t-3' and 5'-aca gtc ttc tga gtg gca gta a-3' (amplicon of 291 bp), as a control. PCR products were electrophoresed in 2% agarose gel, stained with ethidium bromide and photographed using Gel Doc 2000 System (Bio-Rad).
cDNA was synthesized from total RNA of lumbar DRG, brain, spinal cord, liver, kidney and small intestine tissues of healthy rats as above described, and used for multiplex real-time PCR using ABI PRISM 7900HT Sequence Detection Systems and SDS 2.3 software (Applied Biosystems). Primers and probe sets were obtained as TaqMan Gene Expression Assays containing forward and reverse unlabelled PCR primer pair and a fluorescent reporter dye-labelled TaqMan MGB probe (Invitrogen). Samples were analyzed in triplicate in a 10-μl total volume containing 25 ng of cDNA of each tissue, TaqMan universal PCR Master Mix, TaqMan FAM-labelled probes for rat Atp7a gene, Atp7b gene or Ctr1 gene, respectively, and VIC-labelled 18 S ribosomal RNA as endogenous control probe.
The abundance of mRNA of ATP7A, ATP7B, CTR1 or rRNA was measured as the threshold cycle values (Ct) after each reaction. Fluorescence values were plotted against cycle numbers in SigmaPlot 10.0 using sigmoidal 3 parameter fitting and 50% of the maximum fluorescence was taken as the Ct according to Liu at el's method . The relative RNA expression level was calculated using the 2-ρCt method , where gene of interest expression normalized to 18 S rRNA and ρCt = (Ct,ATP7a or ATP7b or CTR1 -Ct,rRNA).
DAB and fluorescent immunohistochemistry of DRG
Animals were euthanized with pentobarbitone and perfused with phosphate buffered saline followed by 4% paraformaldehyde solution. Lumbar 5 DRG was dissected, post-fixated in the perfusion fixative for 2 h, cryoprotected in 30% sucrose overnight and embedded in Tissue-Tek (Sakura Finetechnical, Tokyo, Japan). Cryosections (12 μm) were thaw-mounted onto poly-L-lysine -coated Superfrost plus slides, rinsed, permeabilized in 0.2% Triton X-100, incubated with 1% hydrogen peroxide/methanol mixture (1:1), and blocked in 3% normal goat or donkey serum (Sigma-Aldrich) and 2% BSA (ICPbio Ltd, New Zealand). The slides were incubated with a chicken anti-ATP7A (1:1000; Abcam), a rabbit anti-ATP7B antibody (NB100-360, Novus Biologicals, Littleton, CO, USA) or a rabbit polyclonal anti-hCTR1 antibody (1:500, Novus Biologicals, Littleton, CO, USA), respectively, at room temperature overnight. Following rinses, the slides were incubated subsequently with a biotinylated secondary anti-chicken antibody (1:500, Jackson ImmunoResearch laboratories, PA, USA) or anti-rabbit antibody (1:500, Sigma-Aldrich) for 30 min, followed by an extravidin-peroxidase conjugate (1:500, Sigma-Aldrich) for 30 min. The peroxidase reaction was catalyzed using 3,3'-diaminobenzidine tetrahydrochloride (DAB) (AppliChem, Darmstadt, Germany) and hydrogen peroxide as substrates. The sections were dehydrated by gradient alcohols, cleared in xylene and coverslipped with DPX mounting medium. The negative control sections were processed by excluding the primary antibodies. Digital images were obtained using an Axiocam digital camera attached to an Axiostar light microscope and analyzed using Axiovision 3.0 software on a PC (Carl Zeiss, Hallbergmoos, Germany). For fluorescent double labelling, after blocking, incubation with 200 μl of Invitrogen Image-iT FX signal enhancer for 30 min and washes, DRG sections were incubated with the anti-ATP7A antibody (1:1000, Abcam), anti-hCTR1 antibody (1:1000, Novus) or anti-phosphorylated neurofilament heavy subunit (pNF-H) antibody (1:100, Swant, Bellinzona, Switzerland), respectively, at 4°C for 48 h, followed by subsequently Alexa Fluor 594-labeled anti-chicken or anti-mouse IgG, Alexa Fluro 488-labeled anti-rabbit IgG (1:500, Invitrogen), or DyLight 488-labeled anti-chicken IgG, at room temperature for 3 h. The sections were coverslipped with Vectorshield anti-fade mounting medium (Vector Laboratories, Burlingame, CA, USA). Reciprocal omission controls were included to ensure there was no cross-bleeding between the channels. Digital images were acquired using an Eclipse Ti fluorescence microscope with a cooled colour digital camera attached (Nikon, Japan), and analyzed using Nikon EclipseNet and ImageJ software (National Institutes of Health, USA).
The size profiles of copper transporter-expressing DRG neurons were determined by measuring the staining frequency, mean cell body size and size distribution. Strongly ATP7A-expressing DRG neurons were defined as those having intense diffuse or punctuate cytoplasmic staining and/or plasma membrane immunoreactivity to ATP7A. Those negative for strong ATP7A expression had no or low-intensity diffuse or punctuate cytoplasmic staining without plasma membrane immunoreactivity. Strongly CTR1-expressing DRG neurons were defined as those having intense plasma membrane and/or punctuate cytoplasmic immunoreactivity to CTR1. Those negative for strong CTR1 expression had only diffuse cell body immunoreactivity without plasma membrane or punctuate cytoplasmic immunoreactivity. Between 1,041 and 1,586 neurons from every seventh tissue section were analyzed per DRG per animal for ATP7A and CTR1, respectively. The ATP7A-or CTR1-positive neurons were further arbitrarily categorized into three size-based groups: small ( < 750 μm2), medium (750-1750 μm2) and large ( > 1750 μm2), according to previous studies [26, 61]. To determine the neurotoxicity of oxaliplatin, these morphometric parameters of ATP7A-positive and CTR1-positive subpopulations of DRG neurons were compared between the drug-treated and the control animal groups.
The differences in mean cell body size and staining frequency between different groups were assessed by one-way ANOVA with Bonferroni multiple comparison post test using Prism 5.01 software (GraphPad, San Diego, CA, USA), with a P value of < 0.05 indicating statistical significance.
Linder MC, HazeghAzam M: Copper biochemistry and molecular biology. American Journal of Clinical Nutrition 1996, 63: S797-S811.
Camakaris J, Voskoboinik I, Mercer JF: Molecular mechanisms of copper homeostasis. Biochemical and Biophysical Research Communications 1999, 261: 225–232. 10.1006/bbrc.1999.1073
Lutsenko S, Barnes NL, Bartee MY, Dmitriev OY: Function and regulation of human copper-transporting ATPases. Physiological Reviews 2007, 87: 1011–1046. 10.1152/physrev.00004.2006
Lee J, Pena MMO, Nose Y, Thiele DJ: Biochemical characterization of the human copper transporter Ctr1. Journal of Biological Chemistry 2002, 277: 4380–4387. 10.1074/jbc.M104728200
Barnes N, Tsivkovskii R, Tsivkovskaia N, Lutsenko S: The copper-transporting ATPases, Menkes and Wilson disease proteins, have distinct roles in adult and developing cerebellum. Journal of Biological Chemistry 2005, 280: 9640–9645. 10.1074/jbc.M413840200
Platonova NA, Barabanova SV, Povalikhin RG, Tsymbalenko NV, Danilovskii MA, Voronina OV, Puchkova LV: In vivo expression of copper-transporting proteins in rat brain regions. Biology Bulletin 2005, 32: 108–120. 10.1007/s10525-005-0016-3
Waggoner DJ, Bartnikas TB, Gitlin JD: The role of copper in neurodegenerative disease. Neurobiology of Disease 1999, 6: 221–230. 10.1006/nbdi.1999.0250
Karmy G, Carr PA, Yamamoto T, Chan SHP, Nagy JI: Cytochrome-oxidase immunohistochemistry in rat-brain and dorsal-root ganglia-visualization of enzyme in neuronal perikarya and in parvalbumin-positive neurons. Neuroscience 1991, 40: 825–839. 10.1016/0306-4522(91)90015-G
Rosenfeld J, Cook S, James R: Expression of superoxide dismutase following axotomy. Experimental Neurology 1997, 147: 37–47. 10.1006/exnr.1997.6604
Jeng AY, Fujimoto RA, Chou M, Tan J, Erion MD: Suppression of substance P biosynthesis in sensory neurons of dorsal root ganglion by prodrug esters of potent peptidylglycine alpha-amidating monooxygenase inhibitors. Journal of Biological Chemistry 1997, 272: 14666–14671. 10.1074/jbc.272.23.14666
Zara G, Grassivaro F, Brocadello F, Manara R, Pesenti FF: Case of sensory ataxic ganglionopathy-myelopathy in copper deficiency. Journal of the Neurological Sciences 2009, 277: 184–186. 10.1016/j.jns.2008.10.017
Khaleeli Z, Healy DG, Briddon A, Lunn MP, Reilly MM, Land J, Giovannoni G: Lesson of the Week Copper deficiency as a treatable cause of poor balance. British Medical Journal 2010, 340: 5. 10.1136/bmj.c508
Ishida S, Lee J, Thiele DJ, Herskowitz I: Uptake of the anticancer drug cisplatin mediated by the copper transporter Ctr1 in yeast and mammals. Proceedings of the National Academy of Sciences of the United States of America 2002, 99: 14298–14302. 10.1073/pnas.162491399
Lin XJ, Okuda T, Holzer A, Howell SB: The copper transporter CTR1 regulates cisplatin uptake in Saccharomyces cerevisiae. Molecular Pharmacology 2002, 62: 1154–1159. 10.1124/mol.62.5.1154
Holzer AK, Samimi G, Katano K, Naerdemann W, Lin XJ, Safaei R, Howell SB: The copper influx transporter human copper transport protein 1 regulates the uptake of cisplatin in human ovarian carcinoma cells. Molecular Pharmacology 2004, 66: 817–823. 10.1124/mol.104.001198
Katano K, Safaei R, Samimi G, Holzer A, Rochdi M, Howell SB: The copper export pump ATP7B modulates the cellular pharmacology of carboplatin in ovarian carcinoma cells. Molecular Pharmacology 2003, 64: 466–473. 10.1124/mol.64.2.466
Samimi G, Katano K, Holzer AK, Safaei R, Howell SB: Modulation of the cellular pharmacology of cisplatin and its analogs by the copper exporters ATP7A and ATP7B. Molecular Pharmacology 2004, 66: 25–32. 10.1124/mol.66.1.25
Samimi G, Safaei R, Katano K, Holzer AK, Rochdi M, Tomioka M, Goodman M, Howell SB: Increased expression of the copper efflux transporter ATP7A mediates resistance to cisplatin, carboplatin, and oxaliplatin in ovarian cancer cells. Clinical Cancer Research 2004, 10: 4661–4669. 10.1158/1078-0432.CCR-04-0137
Safaei R, Otani S, Larson BJ, Rasmussen ML, Howell SB: Transport of cisplatin by the copper efflux transporter ATP7B. Molecular Pharmacology 2008, 73: 461–468. 10.1124/mol.107.040980
Kalayda GV, Wagner CH, Buss I, Reedijk J, Jaehde U: Altered localisation of the copper efflux transporters ATP7A and ATP7B associated with cisplatin resistance in human ovarian carcinoma cells. BMC Cancer 2008, 8: 12. 10.1186/1471-2407-8-175
Gregg RW, Molepo JM, Monpetit VJA, Mikael NZ, Redmond D, Gadia M, Stewart DJ: Cisplatin neurotoxicity-The relationship between dosage, time, and platinum concentration in neurologic tissues, and morphological evidence of toxicity. Journal of Clinical Oncology 1992, 10: 795–803.
Screnci D, McKeage MJ, Galettis P: Relationships between hydrophobicity, reactivity, accumulation and peripheral nerve toxicity of a series of platinum drugs. British Journal of Cancer 2000, 82: 966–972. 10.1054/bjoc.1999.1026
Cavaletti G, Tredici G, Pizzini G, Minoia A: Tissue platinum concentrations and cisplatin schedules. Lancet 1990, 336: 1003–1003. 10.1016/0140-6736(90)92462-Q
Krarup-Hansen A, Rietz B, Krarup C, Heydorn K, Rorth M, Schmalbruch H: Histology and platinum content of sensory ganglia and sural nerves in patients treated with cisplatin and carboplatin: an autopsy study. Neuropathology and Applied Neurobiology 1999, 25: 29–40. 10.1046/j.1365-2990.1999.00160.x
McKeage MJ, Hsu T, Screnci D, Haddad G, Baguley BC: Nucleolar damage correlates with neurotoxicity induced by different platinum drugs. British Journal of Cancer 2001, 85: 1219–1225. 10.1054/bjoc.2001.2024
Jamieson SMF, Liu J, Connor B, McKeage MJ: Oxaliplatin causes selective atrophy of a subpopulation of dorsal root ganglion neurons without inducing cell loss. Cancer Chemotherapy and Pharmacology 2005, 56: 391–399. 10.1007/s00280-004-0953-4
Krarup-Hansen A, Helweg-Larsen S, Schmalbruch H, Rorth M, Krarup C: Neuronal involvement in cisplatin neuropathy: prospective clinical and neurophysiological studies. Brain 2007, 130: 1076–1088. 10.1093/brain/awl356
Cavaletti G, Tredici G, Marmiroli P, Petruccioli MG, Barajon I, Fabbrica D: Morphometric study of the sensory neuron and peripheral nerve changes induced by chronic cisplatin (DDP) administration in rats. Acta Neuropathologica 1992, 84: 364–371. 10.1007/BF00227662
Tomiwa K, Nolan C, Cavanagh JB: The effects of cisplatin on rat spinal ganglia - A study by light and electron-microscopy and by morphometry. Acta Neuropathologica 1986, 69: 295–308. 10.1007/BF00688308
Thompson SW, Davis LE, Kornfeld M, Hilgers RD, Standefer JC: Cisplatin neuropathy - Clinical, electrophysiologic, morphologic, and toxicologic studies. Cancer 1984, 54: 1269–1275. 10.1002/1097-0142(19841001)54:7<1269::AID-CNCR2820540707>3.0.CO;2-9
Roelofs RI, Hrushesky W, Rogin J, Rosenberg L: Peripheral sensory neuropathy and cisplatin chemotherapy. Neurology 1984, 34: 934–938.
Cavaletti G, Tredici G, Petruccioli MG, Donde E, Tredici P, Marmiroli P, Minoia C, Ronchi A, Bayssas M, Etienne GG: Effects of different schedules of oxaliplatin treatment on the peripheral nervous system of the rat. European Journal of Cancer 2001, 37: 2457–2463. 10.1016/S0959-8049(01)00300-8
Holmes J, Stanko J, Varchenko M, Ding H, Madden VJ, Bagnell CR, Wyrick SD, Chaney SG: Comparative neurotoxicity of oxaliplatin, cisplatin, and ormaplatin in a Wistar rat model. Toxicological Sciences 1998, 46: 342–351.
Windebank AJ: Chemotherapeutic neuropathy. Current Opinion in Neurology 1999, 12: 565–571. 10.1097/00019052-199910000-00010
Liu JJ, Jamieson SMF, Subramaniam J, Ip V, Jong NN, Mercer JFB, McKeage MJ: Neuronal expression of copper transporter 1 in rat dorsal root ganglia: association with platinum neurotoxicity. Cancer Chemotherapy and Pharmacology 2009, 64: 847–856. 10.1007/s00280-009-1017-6
Muller LJ, Vanderhoop RG, Moorervandelft CM, Gispen WH, Roubos EW: Morphological and electrophysiological study of the effects of cisplatin and ORG.2766 on rat spinal ganglion neurons. Cancer Research 1990, 50: 2437–2442.
Jamieson SMF, Subramaniam J, Liu JJ, Jong NN, Ip V, Connor B, McKeage MJ: Oxaliplatin-induced loss of phosphorylated heavy neurofilament subunit neuronal immunoreactivity in rat DRG tissue. Molecular Pain 2009, 5: 9. 10.1186/1744-8069-5-66
Goss JA, Barshes NR, Karpen SJ, Gao FQ, Wyllie S: Liver ischemia and ischemia-reperfusion induces and trafficks the multi-specific metal transporter Atp7b to bile duct canaliculi: Possible preferential transport of iron into bile. Biological Trace Element Research 2008, 122: 26–41. 10.1007/s12011-007-8057-8
Holzer AK, Varki NM, Le QT, Gibson MA, Naredi P, Howell SB: Expression of the human copper influx transporter 1 in normal and malignant human tissues. Journal of Histochemistry & Cytochemistry 2006, 54: 1041–1049.
El Meskini R, Culotta VC, Mains RE, Eipper BA: Supplying copper to the cuproenzyme peptidylglycine alpha-amidating monooxygenase. Journal of Biological Chemistry 2003, 278: 12278–12284. 10.1074/jbc.M211413200
Hansel DE, May V, Eipper BA, Ronnett GV: Pituitary adenylyl cyclase-activating peptides and alpha-amidation in olfactory neurogenesis and neuronal survival in vitro. Journal of Neuroscience 2001, 21: 4625–4636.
Steveson TC, Ciccotosto GD, Ma XM, Mueller GP, Mains RE, Eipper BA: Menkes protein contributes to the function of peptidylglycine alpha-amidating monooxygenase. Endocrinology 2003, 144: 188–200. 10.1210/en.2002-220716
Wong M, Jeng AY: Posttranslational modification of glycine-extended substance-P by an alpha-amidating enzyme in cultured sensory neurons of dorsal-root-ganglia. Journal of Neuroscience Research 1994, 37: 97–102. 10.1002/jnr.490370113
Erion MD, Tan J, Wong M, Jeng AY: Inhibition of peptidyglycine alpha-amidating monooxygenase by N-substituted homocysteine analogs. Journal of Medicinal Chemistry 1994, 37: 4430–4437. 10.1021/jm00052a002
Hokfelt T, Kellerth JO, Nilsson G, Pernow B: Substance-P-Localization in central nervous-system and in some primary sensory neurons. Science 1975, 190: 889–890. 10.1126/science.242075
McCarthy PW, Lawson SN: Cell type and conduction-velocity of rat primary sensory neurons with substance P-like immunoreactivity. Neuroscience 1989, 28: 745–753. 10.1016/0306-4522(89)90019-5
Lee LW, Prohaska JR, Thiele DJ: Essential role for mammalian copper transporter Ctr1 in copper homeostasis and embryonic development. Proceedings of the National Academy of Sciences of the United States of America 2001, 98: 6842–6847. 10.1073/pnas.111058698
Kim H, Son HY, Bailey SM, Lee J: Deletion of hepatic Ctr1 reveals its function in copper acquisition and compensatory mechanisms for copper homeostasis. American Journal of Physiology-Gastrointestinal and Liver Physiology 2009, 296: G356-G364. 10.1152/ajpgi.90632.2008
Gardiner NJ, Wang Z, Luke C, Gott A, Price SA, Fernyhough P: Expression of hexokinase isoforms in the dorsal root ganglion of the adult rat and effect of experimental diabetes. Brain Research 2007, 1175: 143–154. 10.1016/j.brainres.2007.08.015
Cavaletti G, Fabbrica D, Minoia C, Frattola L, Tredici G: Carboplatin toxic effects on the peripheral nervous system of the rat. Annals of Oncology 1998, 9: 443–447. 10.1023/A:1008231925889
Daugaard GK, Petrera J, Trojaborg W: Eletrophysiological study of the peripheral and central neurotoxic effects of cisplatin. Acta Neurologica Scandinavica 1987, 76: 86–93. 10.1111/j.1600-0404.1987.tb03551.x
Screnci D, Er HM, Hambley TW, Galettis P, Brouwer W, McKeage MJ: Stereoselective peripheral sensory neurotoxicity of diaminocyclohexane platinum enantiomers related to ormaplatin and oxaliplatin. British Journal of Cancer 1997, 76: 502–510.
Harper AA, Lawson SN: Conduction-velocity is related to morphological cell type in rat dorsal-root gangliob neurons. Journal of Physiology-London 1985, 359: 31-&.
Kishi M, Tanabe J, Schmelzer JD, Low PA: Morphometry of dorsal root ganglion in chronic experimental diabetic neuropathy. Diabetes 2002, 51: 819–824. 10.2337/diabetes.51.3.819
Lee KH, Chung KS, Chung JM, Coggeshall RE: Corrlation of cell body size, axon size, and signal conduction-velocity for individually labeled dorsal-root ganglion-cells in the cat. Journal of Comparative Neurology 1986, 243: 335–346. 10.1002/cne.902430305
Holzer AK, Howell SB: The internalization and degradation of human copper transporter 1 following cisplatin exposure. Cancer Research 2006, 66: 10944–10952. 10.1158/0008-5472.CAN-06-1710
Holzer AK, Katano K, Klomp LWJ, Howell SB: Cisplatin rapidly down-regulates its own influx transporter hCTR1 in cultured human ovarian carcinoma cells. Clinical Cancer Research 2004, 10: 6744–6749. 10.1158/1078-0432.CCR-04-0748
Liu JJ, Galettis P, Farr A, Maharaj L, Samarasinha H, McGechan AC, Baguley BC, Bowen RJ, Berners-Price SJ, McKeage MJ: In vitro antitumour and hepatotoxicity profiles of Au(I) and Ag(I) bidentate pyridyl phosphine complexes and relationships to cellular uptake. Journal of Inorganic Biochemistry 2008, 102: 303–310. 10.1016/j.jinorgbio.2007.09.003
Liu WH, Saint DA: Validation of a quantitative method for real time PCR kinetics. Biochemical and Biophysical Research Communications 2002, 294: 347–353. 10.1016/S0006-291X(02)00478-3
Livak KJ, Schmittgen TD: Analysis of relative gene expression data using real-time quantitative PCR and the 2-rrCt method. Methods 2001, 25: 402–408. 10.1006/meth.2001.1262
Bergman E, Ulfhake B: Loss of primary sensory neurons in the very old rat: Neuron number estimates using the disector method and confocal optical sectioning. Journal of Comparative Neurology 1998, 396: 211–222. 10.1002/(SICI)1096-9861(19980629)396:2<211::AID-CNE6>3.0.CO;2-3
This work was supported by a research grant of Cancer Society of New Zealand. V. Ip was a recipient of a PhD scholarship from the Cancer Society of New Zealand.
The authors declare that they have no competing interests.
VI and JL carried out the experimental work. All authors contributed to the research plan, data interpretation and preparation of the manuscript.
Virginia Ip, Johnson J Liu contributed equally to this work.