Received 6 January 2006; Accepted 15 September 2006; Published online: 22 October 2006.

GTP cyclohydrolase and tetrahydrobiopterin regulate pain sensitivity and persistence

Irmgard Tegeder1, 2, 10, Michael Costigan1, 10, Robert S Griffin1, Andrea Abele2, Inna Belfer3, 4, Helmut Schmidt2, Corina Ehnert2, Jemiel Nejim4, 9, Claudiu Marian2, Joachim Scholz1, Tianxia Wu4, Andrew Allchorne1, Luda Diatchenko5, Alexander M Binshtok1, David Goldman3, Jan Adolph2, Swetha Sama5, Steven J Atlas7, William A Carlezon8, Aram Parsegian8, J"orn L"otsch2, Roger B Fillingim6, William Maixner5, Gerd Geisslinger2, Mitchell B Max4 & Clifford J Woolf1

1 Neural Plasticity Research Group, Department of Anesthesia and Critical Care, Massachusetts General Hospital & Harvard Medical School, 149 13th Street, Room 4309, Charlestown, Massachusetts 02129, USA.

2 Pharmazentrum Frankfurt, Institut f邦r Klinische Pharmakologie / Zentrum f邦r Arzneimittelforschung, Entwicklung und Sicherheit, Klinikum der Johann Wolfgang Goethe-Universit"at, Theodor Stern Kai 7, Frankfurt am Main, 60590, Germany.

3 Laboratory of Neurogenetics, National Institute on Alcohol Abuse and Alcoholism, National Institutes of Health, Department of Health and Human Services, 5625 Fishers Lane, Room 3S-32, Rockville, Maryland 20852, USA.

4 National Institute of Dental and Craniofacial Research, National Institutes of Health, Department of Health and Human Services, 10 Center Drive, Building 10, Room 3C-405, Bethesda, Maryland 20892, USA.

5 Center for Neurosensory Disorders, School of Dentistry, 2110 Old Dental Building, CB# 7455 University of North Carolina at Chapel Hill, Chapel Hill, North Carolina 27599-7455, USA.

6 University of Florida College of Dentistry, Community Dentistry and Behavioral Science, 1329 SW 16th Street, Gainesville, Florida 32608, USA.

7 General Medicine Division and the Clinical Epidemiology Unit, Massachusetts General Hospital & Harvard Medical School, 15 Parkman Street, WAC 615, Boston, Massachusetts 02114, USA.

8 Department of Psychiatry, Harvard Medical School, McLean Hospital, 115 Mill Street, Belmont, Massachusetts 02478, USA.

9 Howard Hughes Medical Institute每National Institutes of Health Research Scholars Program, 1 Cloister Court, Building 60, Bethesda, Maryland 20892-1460, USA.

10 These authors contributed equally to this work.

We report that GTP cyclohydrolase (GCH1), the rate-limiting enzyme for tetrahydrobiopterin (BH4) synthesis, is a key modulator of peripheral neuropathic and inflammatory pain. BH4 is an essential cofactor for catecholamine, serotonin and nitric oxide production. After axonal injury, concentrations of BH4 rose in primary sensory neurons, owing to upregulation of GCH1. After peripheral inflammation, BH4 also increased in dorsal root ganglia (DRGs), owing to enhanced GCH1 enzyme activity. Inhibiting this de novo BH4 synthesis in rats attenuated neuropathic and inflammatory pain and prevented nerve injury每evoked excess nitric oxide production in the DRG, whereas administering BH4 intrathecally exacerbated pain. In humans, a haplotype of the GCH1 gene (population frequency 15.4%) was significantly associated with less pain following diskectomy for persistent radicular low back pain. Healthy individuals homozygous for this haplotype exhibited reduced experimental pain sensitivity, and forskolin-stimulated immortalized leukocytes from haplotype carriers upregulated GCH1 less than did controls. BH4 is therefore an intrinsic regulator of pain sensitivity and chronicity, and the GTP cyclohydrolase haplotype is a marker for these traits.

Inflammatory and neuropathic pain result from multiple changes in the peripheral and central nervous systems. Among these are increased excitability and reduced thresholds of primary sensory neurons1, altered spinal cord synaptic processing2, loss of inhibitory interneurons3 and modifications of brainstem input to the spinal cord4. These changes in neuronal activity result from de novo gene transcription, post-translational modifications5, alterations in ion channel conductivity6 and receptor function7, neuroimmune interactions8 and neuronal apoptosis9. Hypersensitivity, manifesting as spontaneous pain, pain in response to normally innocuous stimuli (allodynia) and an exaggerated response to noxious stimuli (hyperalgesia) are the dominant features of clinical pain, and in some individuals they persist long after the initial injury is resolved. It is not understood what perpetuates pain hypersensitivity in only a subset of people. Inbred rodent strain and human twin studies indicate that the risk of developing chronic pain may be genetically determined10, 11, 12.

To reveal genes involved in producing persistent neuropathic pain, we searched the several hundred genes regulated in the dorsal root ganglion (DRG) following sciatic nerve injury13 for those belonging to common metabolic, signaling or biosynthetic pathways and identified upregulation of two of the three enzymes in the synthesis cascade of 6(R)-L-erythro-5,6,7,8-tetrahydrobiopterin (BH4). The regulated enzymes are GTP cyclohydrolase, which catalyzes the first, rate-limiting step, and sepiapterin reductase, which performs the final conversion of 6-pyruvoyltetrahydropterin to BH4 (Fig. 1a).

Figure 1. Regulation of BH4-producing enzymes in the DRG after nerve injury. (a) Biosynthetic pathway of BH4. PTPS, pyruvoyltetrahydropterin synthase. (b,c) Upregulation of BH4-producing enzymes in L4-5 DRG neurons in the SNI model of peripheral neuropathic pain as detected by (b) in situ hybridization 7 d after SNI (b; scale bar 100米m) and by (c) QRT-PCR (c; n = 4, error s.e.m.). ANOVA was consistent with differential expression of GTP cyclohydrolase (GCH1), sepiapterin reductase (SPR) and quinoid dihydropteridine reductase (QDPR) at the indicated time points (*P < 0.05). (d) GCH1 protein expression in L4-5 DRGs after SNI (n = 3, error s.e.m.). (e,f) Neopterin (e) and biopterin (f) concentrations (ng per mg protein) in ipsi- and contralateral L4-5 DRGs 7 d after SNI. DAHP administered 3 h before tissue dissection reduced neopterin and biopterin (n = 6, error s.e.m.). (g) Three days after SNI, GCH1 mRNA每positive neurons are also immunoreactive for the injury-induced transcription factor ATF-3 (scale bar 20米m). *P < 0.05.

BH4 is an essential cofactor for phenylalanine, tyrosine and tryptophan hydroxylases and for nitric oxide synthases. BH4 availability is critical, therefore, for catecholamine, serotonin and nitric oxide synthesis and for phenylalanine metabolism14. Production of BH4 is regulated by GTP cyclohydrolase transcription and activity15, 16. Phosphorylation17, feed-forward activation through phenylalanine18 and feedback inhibition through BH4 tightly regulate GTP cyclohydrolase activity. Mutations in GTP cyclohydrolase or sepiapterin reductase that cause monoamine neurotransmitter deficiency result in DOPA-responsive motor, psychiatric and cognitive disorders19, 20. Given the vital roles of these neurotransmitters, increasing BH4 concentrations may profoundly impact neuronal signaling. We now show that BH4 concentrations are critical for neuropathic and inflammatory pain and that a genetic polymorphism of GTP cyclohydrolase is associated with reduced pain sensitivity and chronicity in humans, owing to reduced BH4 production.

Results

Upregulation of BH4-producing enzymes

We studied the expression of GTP cyclohydrolase, sepiapterin reductase and quinoid dihydropteridine reductase over time in the fourth and fifth lumbar (L4-5) DRGs in the spared nerve injury21 (SNI) model of peripheral neuropathic pain in rats. This model produces long-lasting pain hypersensitivity, including mechanical and cold allodynia. Transcripts of all these enzymes were upregulated (Fig. 1b in situ, Fig. 1c quantitative RT-PCR (QRT-PCR)), with a more than sixfold sustained increase in GTP cyclohydrolase and more modest increases of the other two. Upregulation of GTP cyclohydrolase mRNA was accompanied by increased protein expression (Fig. 1d) and activity (Fig. 1e), the latter indicated by elevated neopterin, an inactive metabolite of dihydroneopterin triphosphate (an intermediate product in the synthesis cascade)22. SNI caused a large elevation of the end product, BH4, as indicated by increases in its stable oxidation product biopterin (Fig. 1f). Double labeling of GTP cyclohydrolase mRNA and the injury-induced nuclear transcription factor ATF-3 (ref. 23) showed that 97 ㊣ 3% of neurons upregulating GTP cyclohydrolase were ATF-3每positive (Fig. 1g). Seven days after SNI, 65 ㊣ 13% of L5 DRG neuronal nuclei expressed ATF-3, reflecting the proportion of cells with axonal damage21. Of these, 75 ㊣ 4% upregulated GTP cyclohydrolase mRNA.

Inhibiting GTP cyclohydrolase reduces neuropathic pain

To test whether increased BH4 synthesis contributes to neuropathic pain, we analyzed the effects of the prototypic GTP cyclohydrolase inhibitor 2,4-diamino-6-hydroxypyrimidine24, 25 (DAHP) in the SNI model.

Injection of a single dose of DAHP (180 mg per kg body weight intraperitoneally (i.p.)) four days after SNI reversed mechanical and cold pain hypersensitivity within 60 min (Fig. 2a). DAHP produced dose-dependent antinociceptive effects on repeated administration without loss of activity (Fig. 2b). It had similar antinociceptive effects when first administered seventeen days after SNI surgery (Fig. 2c), produced analgesia in the chronic constriction injury (CCI) and spinal nerve ligation (SNL) models of peripheral neuropathic pain (Supplementary Fig. 1 online) and was effective on spinal delivery (250米g/kg/h) at 1/30th of the systemic dose (Supplementary Fig. 2 online). The antinociceptive effect of DAHP paralleled the time course of its plasma and CSF concentrations (Fig. 2d), which were within the IC50 range (100每300米M) for GTP cyclohydrolase inhibition in vitro24, 25. DAHP treatment at this dose prevented nerve injury每induced increases in neopterin in the DRG (Fig. 1e), and it significantly reduced biopterin concentrations (Fig. 1f). Biopterin concentrations did not return to preinjury baselines because recycling of BH4 from its oxidation products is not inhibited by DAHP. DAHP (180 mg/kg i.p.) did not change mechanical or heat pain sensitivity in naive animals (Fig. 2e,f) and had no effect on body weight, activity, or performance in the forced swim test26 (Supplementary Fig. 2).

Supplementary Figure 1 (a)Analgesic effects of the GTP cyclohydrolase inhibitor, 2,4-diamino-6-hydroxy-pyrimidine (DAHP) in the chronic constriction injury model (CCI) and (b) in the spinal nerve ligation model (SNL) of peripheral neuropathic pain. DAHP (180 mg/kg i.p.) was injected at the indicated days (n = 9岸10, P < 0.05 for CCI and SNL). Control animals were treated with vehicle. Effect versus time AUCs were used for statistical comparisons of behavioral effects. DAHP effects were significant in both models with P < 0.05. For all panels error bars represent SEM.

Supplementary Figure 2 (a, b) Continuous intrathecal infusion of the GTP cyclohydrolase inhibitor 2,4-diamino-6-hydroxy-pyrimidine (DAHP) reduced mechanical and cold allodynia in the SNI model of neuropathic pain. DAHP(250 ug/kg/h) was delivered to the lumbar spinal cord via a chronically implanted spinal catheter connected to an osmotic Alzet pump. Infusion started right after SNI surgery and continued 14 days, flow rate 5 ul/h (n = 8, P < 0.05). (c) A single intrathecal injection of 6 mg/kg DAHP (arrow) reduced thermal hyperalgesia in the CFA-induced

paw inflammation model (n = 9, P < 0.05). Effect versus time AUCs were used for statistical comparisons. For all panels error bars represent SEM. (d) Effects of DAHP in the Forced Swim Test. Rats (n = 7 per condition) received 3 separate injections of DAHP (180 mg/kg, i.p.), at 1 hr, 19 hrs, and 23 hrs after the first exposure to forced swimming. This commonly used treatment regimen identifies in rats agents with antidepressant or pro-drepressant effects in humans (Mague et al., J Pharmacol Exp Ther. 305:323-30). Retest sessions (forced swim for 300 sec) occurred 24 hr after the first swim exposure and were videotaped from the side of the water cylinders and scored by raters unaware of the treatment condition. Rats were rated at 5 s intervals throughout the duration of the retest session; at each 5 s interval the predominant behavior was assigned to one of four categories: immobility, swimming, climbing, or diving. The totals of these scores are shown for each modality.

Figure 2. Efficacy of DAHP in the spared nerve injury model of neuropathic pain. (a) Injection of DAHP 4 d after SNI (arrow) significantly reduced mechanical (von Frey) and cold (acetone) allodynia (n = 12, P < 0.05). (b) Dose dependent efficacy of DAHP (90, 180 and 270 mg/kg) on mechanical and cold allodynia with repeated daily injections (arrows) in the SNI model, measured 2每3 h after injections (n = 9每10, P < 0.05). The relationship between dose and effect was linear (R = 0.709 and R = 0.754 for mechanical and cold allodynia respectively, P < 0.001). (c) DAHP treatment starting 17 d after nerve injury produced a significant reduction of mechanical and cold pain hypersensitivity (n = 7, P < 0.05). (d) DAHP plasma and CSF concentration time courses after i.p. injection. (e,f) DAHP treatment (arrow) failed to modify mechanical and thermal threshold in naive animals (n = 6, P = 1). For all figures error bars represent s.e.m. The areas under the effect-versus-time curves were used for statistical comparisons of drug effects.

Blocking GTP cyclohydrolase inhibits inflammatory pain

Inflammation produced by hindpaw injection of complete Freund adjuvant (CFA) did not increase GTP cyclohydrolase mRNA expression in the DRG (Supplementary Fig. 3 online). However, intraplantar CFA injection caused significant increases in GTP cyclohydrolase enzyme activity, with increases of neopterin (Fig. 3a) and biopterin (Fig. 3b) in L4-5 DRGs. DAHP (180 mg/kg i.p.) reduced heat hyperalgesia in the inflamed hindpaw (Fig. 3c,d), when administered both before (Fig. 3c) and 24 h after intraplantar (hind paw) CFA injection (Fig. 3d), and normalized neopterin and biopterin concentrations in the DRGs (Fig. 3a,b). Similar efficacy was achieved with intrathecal DAHP (Supplementary Fig. 2; 1/30th systemic dose). DAHP (180 mg/kg i.p.) also reduced flinching behavior in the first and second phases of the formalin test, indicators of acute nociception and activity-dependent central sensitization (Fig. 3e), and this was associated with a reduction in c-FOS每immunoreactive neurons in the dorsal horn of the spinal cord (Fig. 3f,g).

Supplementary Figure 3. Microarray analysis of GTP Cyclohydrolase(GCH1), sepiapterin reductase (SPR) and quinoid dihydropteridine reductase (QDPR) mRNA in ipsilateral lumbar DRGs in the (a) Spared nerve injury model (SNI), (b) the chronic constriction injury model (CCI), (c) the spinal nerve ligation model (SNL) and (d) the complete Freund＊s adjuvant (CFA) induced paw inflammation model. Total RNA was obtained from homogenized tissue by acid phenol extraction (TRIzol reagent, Invitrogen), reverse transcribed using poly-dT as a primer to obtain cDNA fragments. cDNA fragments were amplified by PCR and cloned into the pCR4 vector (TA cloning Kit,Invitrogen). Biotinylated cRNA was produced by in vitro transcription and used for hybridization on the Affymetrix RGU34A chip. Results are means ㊣ SD of triplicate microarrays using pooled tissue of 3 rats per array and time point.

Figure 3. Efficacy of DAHP in inflammatory and formalin-induced pain. (a,b) Neopterin (a) and biopterin (b) concentrations (ng/mg protein) in ipsilateral