Toxicologic Pathology http://tpx.sagepub.com/ The Candidate Neuroprotective Agent Artemin Induces Autonomic Neural Dysplasia without Preventing Peripheral Nerve Dysfunction Brad Bolon, Shuqian Jing, Frank Asuncion, Sheila Scully, Marlese Pisegna, Gwyneth Y. Van, Zheng Hu, Yan Bin Yu, Hosung Min, Ken Wild, Robert D. Rosenfeld, John Tarpley, Josette Carnahan, Diane Duryea, Dave Hill, Steve Kaufman, Xiao-Qiang Yan, Todd Juan, Kathy Christensen, James Mccabe and W. Scott Simonet Toxicol Pathol 2004 32: 275 DOI: 10.1080/01926230490431475 The online version of this article can be found at: http://tpx.sagepub.com/content/32/3/275 Published by: http://www.sagepublications.com On behalf of: Society of Toxicologic Pathology Additional services and information for Toxicologic Pathology can be found at: Email Alerts: http://tpx.sagepub.com/cgi/alerts Subscriptions: http://tpx.sagepub.com/subscriptions Reprints: http://www.sagepub.com/journalsReprints.nav Permissions: http://www.sagepub.com/journalsPermissions.nav Citations: http://tpx.sagepub.com/content/32/3/275.refs.html Downloaded from tpx.sagepub.com by guest on May 24, 2011 Toxicologic Pathology, 32:275–294, 2004 C by the Society of Toxicologic Pathology Copyright  ISSN: 0192-6233 print / 1533-1601 online DOI: 10.1080/01926230490431475 The Candidate Neuroprotective Agent Artemin Induces Autonomic Neural Dysplasia without Preventing Peripheral Nerve Dysfunction BRAD BOLON,1 SHUQIAN JING,2 FRANK ASUNCION,3 SHEILA SCULLY,1 MARLESE PISEGNA,3 GWYNETH Y. VAN,1 ZHENG HU,2 YAN BIN YU,2 HOSUNG MIN,4 KEN WILD,5 ROBERT D. ROSENFELD,6 JOHN TARPLEY,1 JOSETTE CARNAHAN,5 DIANE DURYEA,1 DAVE HILL,1 STEVE KAUFMAN,1 XIAO-QIANG YAN,1 TODD JUAN,1 KATHY CHRISTENSEN,1 JAMES MCCABE,1 AND W. SCOTT SIMONET2 Departments of 1 Pathology, 2 Exploratory Biology, 3 Inflammation, 4 Functional Genomics, 5 Neurobiology and 6 Protein Chemistry, Amgen Inc., Thousand Oaks, California 91320-1799, USA ABSTRACT Artemin (ART) signals through the GFRα–3/RET receptor complex to support sympathetic neuron development. Here we show that ART also influences autonomic elements in adrenal medulla and enteric and pelvic ganglia. Transgenic mice over-expressing Art throughout development exhibited systemic autonomic neural lesions including fusion of adrenal medullae with adjacent paraganglia, adrenal medullary dysplasia, and marked enlargement of sympathetic (superior cervical and sympathetic chain ganglia) and parasympathetic (enteric, pelvic) ganglia. Changes began by gestational day 12.5 and formed progressively larger masses during adulthood. Art supplementation in wild type adult mice by administering recombinant protein or an Art-bearing retroviral vector resulted in hyperplasia or neuronal metaplasia at the adrenal corticomedullary junction. Expression data revealed that Gfrα–3 is expressed during development in the adrenal medulla, sensory and autonomic ganglia and their projections, while Art is found in contiguous mesenchymal domains (especially skeleton) and in certain nerves. Intrathecal Art therapy did not reduce hypalgesia in rats following nerve ligation. These data (1) confirm that ART acts as a differentiation factor for autonomic (chiefly sympathoadrenal but also parasympathetic) neurons, (2) suggest a role for ART overexpression in the genesis of pheochromocytomas and paragangliomas, and (3) indicate that ART is not a suitable therapy for peripheral neuropathy. Keywords. Artemin; GFRα3; adrenal medulla; neuropathy; paraganglioma; pheochromocytoma; sympathetic chain. INTRODUCTION Glial cell line-derived neurotrophic factor (GDNF)1 is the prototypic member (Lin et al., 1993) of a novel class of neurotrophic factors with structural similarities to the transforming growth factor β (TGFβ) superfamily. Other ligands of the GDNF family include neurturin (NTN) (Kotzbauer et al., 1996), persephin (PSP) (Milbrandt et al., 1998), and artemin (ART) (Baloh et al., 1998; Masure et al., 1999; Rosenblad et al., 2000). These factors act, either alone or in series, to promote survival of diverse neuronal groups in both the central (CNS) and peripheral nervous (PNS) systems (Lin et al., 1993; Buj-Bello et al., 1995; Baloh et al., 1998; Horger et al., 1998; Milbrandt et al., 1998; Akerud et al., 1999; Forgie et al., 1999; Fundin et al., 1999; Heuckeroth et al., 1999; Baudet, et al., 2000; Enomoto et al., 2000; Rosenblad et al., 2000). GDNF is also an essential morphogen for early development of both the kidneys and the enteric nervous system (Moore et al., 1996; Pichel et al., 1996; Sanchez et al., 1996), and possibly of craniofacial structures and organ positioning (Homma et al., 2000). Both GDNF and NTN also participate in differentiation of cutaneous sensory innervation (Fundin et al., 1999) and hair cycle control (Botchkareva et al., 2000), while GDNF also specifies spermatogonial fate (Meng et al., 2000). Biological properties of the GDNF ligand family have been thoroughly reviewed in recent publications (Saarma and Sariola, 1999; Baloh et al., 2000). The GDNF family ligands signal through a complex receptor consisting of 2 subunits, an extracellular glycosylphosphatydlinositol (GPI)-linked GDNF family receptor-alpha (GFRα) receptor (Jing et al., 1996; Treanor et al., 1996; Baloh et al., 1997; Jing et al., 1997; Sanicola et al., 1997) and an intracellular RET receptor protein tyrosine kinase (Takahashi et al., 1988). Four GFRα proteins have been demonstrated in mammalian tissues: GFRα-1 (Jing et al., 1996; Treanor et al., 1996), GFRα–2 (Baloh et al., 1997; Buj-Bello et al., 1997; Jing et al., 1997; Klein, R.D. et al., 1997; Sanicola et al., 1997), GFRα–3 (Jing et al., 1997; Baloh et al., 1998; Naveilhan et al., 1998; Trupp et al., 1998), and GFRα–4 (Lindahl et al., 2000). In vitro and in vivo studies indicate that each GFRα exhibits high affinity binding and potent RET activation (i.e., is “specific”) when paired with a single trophic factor (Jing et al., 1996; Treanor et al., 1996; Baloh et al., 1997; Klein et al., 1997; Baloh et al., 1998; Leitner et al., 1999; Masure et al., 1999; Masure et al., 2000; Lindahl Address correspondence to: Dr. Scott Simonet, Amgen, One Amgen Center Drive, M/S 14-1-B, Thousand Oaks, CA 91320-1799, USA; e-mail: ssimonet@amgen.com 1 Abbreviations: ANS, autonomic nervous system; ApoE, apolipoprotein E; ART/Art, artemin; ChAT, choline acetyltransferase; CHO, Chinese hamster ovary (cell line); CNS, central nervous system; DRG, dorsal root ganglia; E, gestational day; GDNF/Gdnf, glial-derived neurotrophic factor; GFAP, glial fibrillary acidic protein; GFRα/Gfrα, GDNF family receptor type alpha; GPI, glycosylphosphatydlinositol; MEN, multiple endocrine neoplasia; NFP, neurofilament protein; NTN/Ntn, neuturin; OPG, osteoprotegerin; P, postnatal day; PGP, protein gene product; PNS, peripheral nervous system; PSP/Psp, persephin; RET/Ret, RET (REarranged during Transfection) receptor tyrosine kinase; SCG, superior (cranial) cervical ganglion; TGFβ, transforming growth factor beta; TH, tyrosine hydroxylase. 275 Downloaded from tpx.sagepub.com by guest on May 24, 2011 276 BOLON ET AL. et al., 2001). The preferred interactions are GDNF-GFRα–1, NTN-GFRα–2, ART-GFRα–3, and PSP-GFRα–4. Alternative pairings (GDNF-GFRα–2, NTN-GFRα–1) are also functional in vitro ((Baloh et al., 1997; Buj-Bello et al., 1997; Jing et al., 1997; Klein et al., 1997; Sanicola et al., 1997; Cacalano et al., 1998), although the significance of such interactions in vivo is not known. An alternative interaction for ART with GFRα–1 has been proposed (Baloh et al., 1998; Rosenblad et al., 2000), but this pairing is controversial (Masure et al., 1999). Biological functions for the high affinity interactions of GDNF-like ligands and their respective GFRs have been elucidated by gene targeting studies. Mice with null mutations of either Gdnf (Moore et al., 1996; Pichel et al., 1996; Sanchez et al., 1996) or Gfrα–1 (Cacalano et al., 1998; Enomoto et al., 1998) die soon after birth as a consequence of enteric neuronal and renal agenesis. In contrast, mice lacking Ntn (Heuckeroth et al., 1999) or Gfrα–2 (Rossi et al., 1999) are viable and exhibit no substantive gross defects, although microscopic examination reveals a decreased number of myenteric (parasympathetic) ganglia and deficits in selected populations of sensory neurons. The only macroscopic phenotype is ptosis (drooping of the eyelids), resulting from a lack of parasympathetic innervation to structures in this region. In like manner, deletion of Art or Gfrα–3 yields viable animals with ptosis, although in these instances the neuronal deficit resides in abnormal sympathetic innervation subsequent to aberrant development of the superior (cranial) cervical ganglion (SCG) (Nishino et al., 1999; Honma et al., 2002). Mice with null mutations of Art or Gfrα–3 also exhibit widespread migration defects in the peripheral sympathetic nervous system (Honma et al., 2002). GDNF-like ligands have been proposed as agents to ameliorate central neurodegenerative diseases. For example, GDNF appears to selectively preserve midbrain dopaminergic neurons of the nigrostriatal pathway (Hoffer et al., 1994; Beck et al., 1995; Tomac et al., 1995; Hou et al., 1996; Rosenblad et al., 2000). In like manner, NTN has a similar impact on nigral neuron survival, although it may be less effective than GDNF in supporting functional maturity of neurons (Akerud et al., 1999). Both GDNF and NTN also support parasympathetic neurons (Hashino et al., 2001). ART exhibits developmental stage-specific enhancement of sympathetic neuron survival both in utero and after birth (Andres et al., 2001), serving particularly as an early mediator of enteric innervation with sympathetic neurons (Enomoto et al., 2001). Administration of ART has been postulated as a treatment for peripheral neuropathies based on the localization of ART/GFRα–3 to the PNS rather than the CNS (Baloh et al., 1998; Masure et al., 1999)—particularly those relevant to nociceptive sensory pathways (Orozco et al., 2001)—and the upregulation of ART expression in the distal segment of transected sciatic nerves (Baloh et al., 1998). The present experiments were performed to test this hypothetical efficacy as well as to clarify discrepancies between prior descriptions of ART biology. Our data did not provide evidence to support the use of ART as a neuroregenerative agent. Instead, our findings indicate that therapy with ART can precipitate autonomic neural expansion in adult animals, suggesting that undesirable side effects will outweigh the potential benefits afforded by ART. TOXICOLOGIC PATHOLOGY METHODS Approval: This study was conducted in accordance with federal animal care guidelines and was preapproved by the Amgen Institutional Animal Care and Use Committee. Cloning of Mouse Art and Human ART: An expressed sequence tag (designated Gdnf-related neurotrophic factor 4 [Grnf4]) with 47% homology to the C-terminal domain of Ntn was isolated from a bone cDNA library derived from crushed osteoporotic femurs and tibias of four 6-week-old, female B6D2F1/CrlBR mice (acquired by crossing C57BL/6NCrlBR females with DBA/2NCrlBR males) with null mutations for osteoprotegerin (Opg; (Simonet et al., 1997)). Cloning of full-length murine Art and human ART cDNAs by rapid amplification of cDNA ends (5’ RACE) revealed open reading frames encoding molecules of 224 and 228 amino acids, respectively. At the amino acid level, the homology between these two molecules was 77%. During our characterization of Grnf4, the protein was described by three independent labs as ART (Baloh et al., 1998), enovin (Masure et al., 1999) and neublastin (Rosenblad et al., 2000). Our human GRNF4 and mouse Grnf4 clones were identical to the published ART sequences for the gene and protein. Thus, we have adopted the ART nomenclature for the remainder of this report. [125 I]Art Binding Assays: Recombinant mouse Art (mArt) was produced and purified from E. coli and mammalian (CHO cell) expression systems. Neuro-2a cells (ATCC #CCL 131), a mouse neuroblastoma cell line, were transfected with a pBK RSV plasmid containing mouse Gfrα–3 cDNA (cloned as described previously; Jing et al., 1997). Three clones (NSR-1, NGR-5, and NGR-19) resistant to G418 (Sigma, St. Louis, MO) were expanded and assayed for Gfrα–3 expression by Northern blot using Gfrα–3 cDNA probes and by binding (Jing et al., 1996) to [125 I]Art (custom iodination; Amersham, Arlington Heights, IL). Chemical cross-linking of [125 I]Art to Gfrα–3 and Ret expressed in NSR-5 cells or a Gfrα–3/hFc fusion protein (mouse Gfrα–3, fused in-frame with the Fc region of human IgG1 (Culouscou et al., 1995)) was performed as described (Jing et al., 1996, 1997). Art-induced Ret autophosphorylation was examined by immunoblot analysis as described previously (Jing et al., 1996). Briefly, NSR-5 cells were treated with recombinant Art and lysed. The lysates were immunoprecipitated with an anti-Ret antibody, fractionated by SDS-PAGE, transferred onto nitrocellulose filters, and probed with an anti-phosphotyrosine antibody (Upstate Biotechnology, Lake Placid, NY). As appropriate, experiments employed either control neuro-2a cells lacking Gfrα or engineered to express Gfrα–1 (NGR-38 line; (Jing et al., 1996)) or Gfrα–2 (NNR-9 line), or other soluble receptors (Gfrα–1/hFc or Gfrα–2/hFc; (Jing et al., 1996)). ART Activity in In Vitro Neuronal Survival Assays Primary cultures of mouse neural cells incorporating mixed neuronal and glial populations were performed using standard methods (Patterson and Chun, 1977; Hawrot and Patterson, 1979; Carnahan and Patterson, 1991). Briefly, dorsal root (DRG), enteric, and superior cervical (SCG) ganglia were harvested from B6D2F1/CrlBR mice and Crl:CD (SD)IGS BR rats at various times during gestation (E15, E16) Downloaded from tpx.sagepub.com by guest on May 24, 2011 Vol. 32, No. 3, 2004 ARTEMIN INDUCES NEURAL PROLIFERATION or early postnatal (P0, P7) development. Cells were collected in L15-air medium, dissociated with collagenase accompanied by repeated, gentle pipetting, and seeded on collagencoated 96-well plates (1x106 cells/well). Cultures were grown for 2 days in L15-C02 medium supplemented with 10 ng/ml of nerve growth factor (NGF), after which they were switched to medium containing neutralizing anti-NGF antibodies plus different concentrations of Gdnf, Ntn, Art, Psp, or no other factors. Assays were conducted in triplicate for each factor tested. Effects of growth factors on neuronal survival were measured after two days by counting neurite-bearing (i.e., live) cells by phase-contrast microscopy. Expression of ART and GFRα–3: Northern blot analysis for ART expression in adult organs was performed in batteries of human and mouse tissues that were comparable to those tested previously (Baloh et al., 1998), except that our assessment also included peripheral blood leukocytes. Northern analysis was performed on human multiple tissue Northern blot, human multiple tissue Northern blot II, and mouse multiple tissue Northern blot (all from Clontech Laboratories, Palo Alto, CA) and probed with 32 P-labeled DNA derived from either the human ART open reading frame (nucleotides 484-672) or mouse Art open reading frame (nucleotides 649-954). Radiolabeling of probes was performed using RediPrime II (Amersham Biosciences), and hybridization was carried out in ExpressHyb solution (Clontech). Signal was detected using a Phosphor Imager (Molecular Dynamics). In addition, tissue-specific distributions of Art and Gfrα–3, and for some tissues other Gfrα and Ret, were assessed in wild-type mice using intact embryos (E10.5, E11.5, E12.5, E13.5), fetuses (E15.5, E18.5), and selected adult tissues. Specimens were fixed by immersion in zinc formalin (Z-Fix; Anatech Ltd., Battle Creek, MI), processed into paraffin, sectioned serially at 4 µm, and hybridized to 33 P-labeled riboprobes using standard methods (Wilkinson, 1993). The probe sequences used for Gfrα–1, Gfrα–2, Gfrα–3, and Ret have been published previously (Yu et al., 1998); the sequence of the probe for mouse Art encompassed nucleotides 413-867 (Gb: AAA46613) of the open reading frames. In selected tissues, the pattern of Art expression was confirmed by a ribonuclease protection assay and cDNA probes (using sequences comparable to those of the riboprobes). In addition, the distribution of Gfrα–3 was investigated in Gfrα–3 null mutant mouse embryos (E9.5, E10.5, E12.5) using enzyme histochemistry (Mercer 1995) to detect the Gfrα–3/lacZ fusion protein (see next). Targeted Disruption of Gfrα–3: A 3.5 kb EcoR I and Xho I DNA fragment containing exon 1 (encoding amino acids 1-27) and a 10.0 kb EcoR V and Kpn I DNA fragment containing exon 2 (encoding amino acids 28-123) and exon 3 (encoding amino acids 124–154) of mGfrα–3 were isolated from a 129/SvJ mouse BAC genomic DNA library (Genome Systems, St. Louis, MO). Two oligonucleotide primers (5′ -CGG CTC GAG AAG CTT CAT GGC GGG TGG ACG C-3′ , 5′ -CGC CTC GAG AGT GCT GGG ATT AAA GAC ATG TGC-3′ ) were used for PCR amplification of a 3.2 kb fragment containing the “translation starting” codon and upstream sequence of the Gfrα–3 gene. The PCR fragment was fused in-frame to the 5′ -end of a lacZ/neo-cassette included in the pBlueScript (Stratagene, La Jolla, CA) derived-plasmid 277 pJH17 (Figure 1). A 3.8 kb Sca I fragment from the 10.0 kb genomic fragment was introduced to the 3′ -end of the neo cassette of the same plasmid to generate the targeting construct pHJ80 (Figure 1). Embryonic stem (ES) cells derived from a 129/SvJ mouse embryo (RW4 line) were transfected with linearized pJH80, and six G418-resistant ES clones containing an 11 kb deletion (encoding amino acids 2 to 123) of Gfrα–3 were injected into C57BL/6J blastocysts. Chimeric (agouti) offspring were mated to Tac:N:NIH(S)-BC (Black Swiss) mice at 6–8 weeks of age. Germline transmission of the mutant allele was assessed by Southern and/or PCR analyses. For PCR genotyping of potential founders, genomic DNA was amplified using primers based on the following sequences (Figure 1): 1, a forward primer based on an upstream intron sequence of Gfrα–3 exon 1 (5′ - ACA GTA GGT GGG CAG ACT CTA GTG G-3′ ); 2, a reverse primer based on the lacZ gene sequence (5′ -AGT CAC GAC GTT GTA AAA CGA CGG-3′ ); and 3, a reverse primer based on the downstream intron sequence of exon 1 that was deleted in the mutant allele (5′ -CAA GCC TCT CTG TAG CAA GTC TAC G-3′ ). Absence of Gfrα–3 expression in homozygous knockout mice was confirmed by RT-PCR using total cellular RNA and primers (not shown) based on the selected Gfrα–3 sequences: 1, a forward primer based on the deleted portion of exon 2 (5′ -GAA ACT CCC TTC CCA CAG AGA AC-3′ ); 2, a forward primer found in exon 3, (5′ -GTG ACT ACG AGT TGG ACG TCT C-3′ ); and 3, a reverse primer based on the sequence downstream from exon 3 (5′ -GAG GAT GTC CAT AGG GTG GCA G-3′ ). Construction of Art-Transgenic Mice: As shown in Figure 2, the full coding region of mouse Art was subcloned into 2 independent expression vectors, placing it under the control of either the human ß-actin promoter and enhancer for ubiquitous expression (Klebig et al., 1995), or under the control of the human apolipoprotein E (ApoE) promoter and liver specific enhancer (Simonet et al., 1994). Single-cell B6D2F1/CrlBR mouse embryos were injected with 1 transgene as described (Brinster et al., 1985). Transgenic offspring from 2 lines per construct were identified by screening for SV40 poly A in genomic DNA prepared from ear punch biopsies (Simonet et al., 1994). Expression analysis of the ß-actin Art transgene was performed by RT-PCR using total cellular RNA from spleen and oligomer probes for sequences in the ß-actin promoter (5′ -AGC ACA GAG CCT CGC CTT TGC CGA TC-3′ ) and the 3′ Art open reading frame (5′ -GCG GGA CAT TGG GTC CAG GGA AGC–3′ ). Expression of the ApoE Art transgene was done via northern blot analysis in total cellular RNA from liver and the Art cDNA probe. In a follow-up experiment, lethally irradiated adult (16- to 18-weeks-old) female B6D2F1/CrlBR mice were injected IV with bone marrow cells that had been transfected with a retroviral expression vector containing Art cDNA under the control of the retroviral promoter, or with vector alone, according to standard methods (Yan et al., 1995). Expression of Art mRNA was assessed after 8 weeks in bone marrow and spleen cells acquired at necropsy by northern blot analysis using total cellular RNA and Art cDNA probes. Bioassays for Activity of Artemin: The function of Art was tested in vivo by 3 experiments. In the first bioassay, developing (E12, E14, E17) and young adult (2–3 months Downloaded from tpx.sagepub.com by guest on May 24, 2011 278 BOLON ET AL. TOXICOLOGIC PATHOLOGY FIGURE 1.—Generation of Gfrα–3 null mutant mice. Schematic representation of the Gfrα–3 gene targeting vector (top), the wild-type Gfrα–3 locus (middle), and the expected mutant allele (bottom). In the targeting construct, a stator-less LacZ-neo cassette was fused in frame to the starting codon (ATG) of Gfrα–3 exon 1. Homologous recombination of the targeting construct with the endogenous allele resulted in deletion of an 11 kb segment of the Gfrα–3 gene that included the first 2 exons (coding for amino acids 2 to 123). [The 3 exons (Ex) are depicted as white boxes.] The location and size of the DNA fragments (generated by Hind III or Kpn I digestion) before and after homologous recombination are shown beneath the alleles. The arrowheads indicate the placement and direction of transcription for the three PCR genotyping primers. old) Art-transgenic mice for both the β-actin and ApoE constructs were analyzed to define any phenotype resulting from chronic overexpression of ART throughout development. In the second model, two-month-old wild type C57BL/6J mice of both sexes received subcutaneous injections of either 0 (n = 3 to 4 per sex per genotype) or 5 (n = 4 to 5 per sex per genotype) mg of Art 119-224/kg in PBS for 14 consecutive days. One hour prior to necropsy, each adult FIGURE 2.—Generation of Art-transgenic mice. Schematic representation of the Art transgene in relationship to the apoplipoprotein E (ApoE, top) and β-actin (bottom) promoters and various restriction sites. Exon I and intron I are specific for their respective promoters. The SV-40-derived DNA segment denoted by dark bars beneath the constructs were used as probes for both genotyping (PCR) and expression (Northern) analyses. Abbreviations: HCR = hepatic control region, SV40-PA = simian virus 40 polyamine sequence. Downloaded from tpx.sagepub.com by guest on May 24, 2011 Vol. 32, No. 3, 2004 ARTEMIN INDUCES NEURAL PROLIFERATION mouse was injected IP with 50 mg 5-bromo-2′ -deoxyuridine (BrdU)/kg (Sigma). Animals were anesthetized with isoflurane, and blood was drawn by cardiac puncture for clinical pathology analyses. Major organs were examined for gross and microscopic lesions. Serial 4-µm-thick sections of neural tissues were stained by standard indirect immunohistochemistry to detect proliferating cells (rat monoclonal anti-BrdU 1:400; Accurate, Westbury, NY), neurons (rabbit polyclonal anti-human protein gene product 9.5 [PGP 9.5] 1:500; Accurate (Wilkinson et al., 1989)), parasympathetic neurons (goat polyclonal anti-choline acetyltransferase [ChAT] 1:40; Chemicon, Temecula, CA (Nishino et al., 1999)), sympathetic neurons (rabbit polyclonal anti-tyrosine hydroxylase [TH] 1:100; Chemicon (Nishino et al., 1999)), or glia (rabbit polyclonal anti-glial fibrillary acidic protein [GFAP] 1:400; Dako, Carpenteria, CA). Primary antibodies were detected using biotinylated secondaries made against the species of the primary antibody (Vector Labs, Burlingame, CA or Jackson ImmunoResearch, West Grove, PA) followed by a Vector Elite ABC kit (Vector Labs, used according to the manufacturer’s instructions); diaminobenzidine tetrachloride (DAB; Sigma) as the chromagen; and hematoxylin as the counterstain. In the third bioassay, the therapeutic potential of Art with respect to peripheral neuropathy was tested in young adult (200 grams) male Crl:CD (SD)IGS BR rats using a surgical ligation model of neuropathic pain (Kim and Chung, 1992). Animals were anesthetized with isoflurane, and the lumbar spinal nerve roots at the level of L5 and L6 were tightly ligated between the DRG and the sciatic nerve to induce mechanical (tactile) allodynia in the left hind paw. Half of the rats also received a chronically indwelling catheter in the intrathecal space near the lumbar intumescence. After at least seven days of recovery, allodynia was assessed by recording the pressure at which the left hind paw was withdrawn from graded tactile stimuli (imposed by von Frey filaments ranging from 4.0 to 148.1 mN) that were applied perpendicular to the plantar surface between the footpads. A paw withdrawal threshold (PWT) was determined by sequentially increasing and decreasing the stimulus strength and analyzing withdrawal data using a Dixon non-parametric test (Chaplan et al., 1994). Normal and sham-operated (i.e., nerves isolated but not ligated) control rats withstand at least 148.1 mN (equivalent to 15 g) of pressure without responding, while animals with ligated spinal nerves exhibit PWT as low as 4.0 mN (equivalent to 0.41 g). In the present study, rats that did not exhibit motor dysfunction (e.g., paw dragging or dropping) but had PWT reduced below 39.2 mN (equivalent to 4.0 g) were treated with Art 119-224 or vehicle twice daily for 5 days by either IP injection (5 mg/kg) or intrathecal (IT) administration (10 µg unit dose). PWT were acquired at 10, 20, 30, 40, 50, and 60 minutes after the initial dose and once per day on treatment days 2 through 5. RESULTS Details concerning the post-translational processing of mouse Art and human ART have been described previously (Baloh et al., 1998; Masure et al., 1999; Rosenblad et al., 2000). However, our in vitro and in vivo experiments provide novel data that expands upon and clarifies the prior findings relevant to the biology of ART signaling. 279 Two Secreted Art Variants Originate by Processing at Alternate Cleavage Sites Expression of full-length murine Art in mammalian (CHO) cells revealed that secreted versions of mature Art were released into the conditioned media (Figure 3). An SDSPAGE gel silver stain analysis of purified Art from CHO cells run under non-reducing and reducing conditions revealed that Art migrated as 2 bands with apparent molecular weights of 34 kDa and 17 kDa, respectively (Figure 3, lanes 2 and 5), thus indicating that Art exists as a disulfidelinked dimer in solution. The presence of N -linked oligosaccharides on mammalian Art was indicated by the capacity for N -glycanase treatment to reduce these bands to approximately 24 kDa and 12 kDa, respectively, under either nonreducing as well as reducing conditions (Figure 3, Lanes 3 and 6). Microsequence analysis obtained from purified mammalian cell-derived material revealed that these 2 species of secreted Art resulted from cleavage between amino acids 111 and 112—the principal location (Baloh et al., 1998), found immediately adjacent to an RXXR proteolytic cleavage site—and at position 120 and 121 (Figure 3). Thus, processing of pre-pro Art in CHO cells consistently yielded a mixture of 2 mature proteins, a predominant molecule with 113 amino acids (Art 112–224) intermingled with a less abundant 104 amino acid form (Art 121-224); an intermediate 106 amino acid version (Art 119-224) produced by expression of the gene in an E. coli system was not found in the CHOderived product. Analysis of secreted human ART indicates that comparable posttranslational processing occurs, and that the upstream site yielding a 113 aa peptide is also the predominant one used for posttranslational processing (data not shown). Art Binds Only to Gfrα–3 Specific binding of Art to Gfrα–3 but not Gfrα–1 was confirmed by chemical cross-linking experiments. When [125 I]Art (either the CHO-derived 112–224 or E. coliderived 119–224 forms) was incubated with a soluble Gfrα receptor, strong bands of ∼85 kD, ∼180 kD, and ∼360 kD were detected only for the Gfrα–3/hFc fusion protein (Figure 4A). No cross-linked products were observed if Gfrα–1/hFc or Gfrα–2/hFc were used, or if unlabeled Art was added. In the control experiment, binding of [125 I]Gdnf to Gfrα–1/hFc or [125 I]Ntn to Gfrα–2/hFc could be blocked using an excess of unlabeled ligand but was unaffected by the addition of Art (Figure 4A). In like manner, association of [125 I]Art with Ret occurred over a wide range of ligand concentrations (data not shown) but only in cells that expressed Gfrα–3 (lines NSR-1, NSR-5 [Figure 4B], and NSR-19). Prominent bands developed at ∼95 kD, ∼130–170 kD, and ∼190 kD, with less intense bands apparent at ∼48 kD, ∼60 kD, and ∼380 kD as well as a faint band at a very high molecular weight position. Formation of these bands was effectively inhibited by addition of unlabeled Art but was not affected by the addition of Gdnf. Under the same conditions, bands were not observed when [125 I]Art was incubated with control cells expressing Gfrα–1 (line NGR-38) or Gfrα–2 (line NNR-9), even at a concentration of 1 nM (Figure 4B). Downloaded from tpx.sagepub.com by guest on May 24, 2011 280 BOLON ET AL. TOXICOLOGIC PATHOLOGY FIGURE 3.—Posttranslational processing of Art. Screening of E. coli- and CHO cell-derived recombinant mouse Art to define posttranslational modifications to the mature protein; 12% SDS-PAGE gel stained with silver. Proteins run under nonreducing (Lanes 1 to 4) and reducing (Lanes 5 to 7) conditions included E. coli-derived rMet-Art 119-224, 0.25 µg (Lanes 1 and 7) and CHO-derived Art (present as both 112-224 and 121-224 forms), 0.6 µg, without (Lanes 2 and 5) and with (Lanes 3 and 6) N -glyconase. Lane 4 was loaded with a carboxymethylated molecular weight standard (Pharmacia) (Lane 4). The laddering pattern confirms that the 2 secreted forms of mature mammalian Art are disulfide-linked, N -glycosylated dimers. Downloaded from tpx.sagepub.com by guest on May 24, 2011 Vol. 32, No. 3, 2004 ARTEMIN INDUCES NEURAL PROLIFERATION 281 FIGURE 4.—Interaction of Art with Gfrα receptors and the Ret tyrosine kinase. In vitro studies were performed with mouse Art derived from both E. coli (119-224) and CHO cell (112-224; data not shown) expression systems. Binding of [125 I]Art was assessed by chemical cross-linking to fusion proteins incorporating a human immunoglobulin constant domain with a mouse Gfrα (A) or to neuro-2a mouse neuroblastoma cells transfected with either mouse Gfrα–1 (NGR-38 line), Gfrα–2 (NNR-9 line) or Gfrα–3 (NSR-5 line) (B). Art was bound only to Gfrα–3 fusion protein (A) or cells expressing Gfrα–3 (B). Excess unlabeled Art blocked the interaction of [125 I]Art with Gfrα–3 but had no effect on binding of [125 I]Gdnf with Gfrα–1 or [125 I]Ntn with Gfrα–2 (A). Ret activation by GDNF family ligands was examined in lysates of cells expressing a single Gfrα and Ret (C). In the presence of Art, Ret was autophosphorylated only in cells expressing Gfrα–3 (NSR-1 and NSR-5 lines). Neither Gdnf nor Ntn induced Ret autophosphorylation via Gfrα–3, although these ligands were effective in control cells that expressed Gfrα–1 (NGR-38 line) or Gfrα–2 (NNR-5 line), respectively. Psp did not produce Ret autophosphorylation in cells expressing any of these three Gfrα. Downloaded from tpx.sagepub.com by guest on May 24, 2011 282 BOLON ET AL. Art Signaling Occurs Only through Gfrα–3 Treatment of Gfrα–3 expressing cells with Art activated signal transduction as indicated by induction of tyrosine autophosphorylation on the mature cell surface form of Ret (Figure 4C). This finding was apparent as a strong ∼170 kD band on immunoblots developed with an antiphosphotyrosine antibody. In control samples, Gdnf and Ntn induced Ret autophosphorylation in cells expressing Gfrα–1 or Gfrα–2, respectively (Figure 4C). However, these two ligands could not stimulate Ret autophosphorylation in Gfrα–3 expressing cells (Figure 4C). Ret autophosphorylation was not affected by Psp in cells bearing Gfrα–1, Gfrα–2, or Gfrα–3 (Figure 4C). The induction of Ret autophosphorylation by the Art/ Gfrα–3 complex was dose-dependent (data not shown). Art activity was detected at 2 pM; the pathway was saturated at 200 pM. Very strong Ret autophosphorylation following addition of Art was observed within 1 minute and was maximal at 10 minutes (data not shown). TOXICOLOGIC PATHOLOGY Art Is a Neurotrophic Factor for Sympathetic and Parasympathetic Neurons Our in vitro data confirmed the previous report (Baloh et al., 1998) that ART exhibits a trophic action toward neurons in several peripheral ganglia while adding significant new information regarding the biology of the Art/Gfrα–3 signaling pathway. For example, Art effectively supported sensory neuron survival in rat DRG at and after birth (Figure 5A) but not at E16 (data not shown). Both E coli- and CHO-cell derived Art 112–224 exhibited comparable efficacy, indicating that posttranslational modification was not required for activity (Figure 5A). Art 112–224 was approximately 2-fold more effective in rescuing neurons (Figure 5A). In contrast, Art did not support neuronal survival in DRG of neonatal Gfrα–3 null mutant mice (Figure 5B), thus providing additional confirmation that Art signaling requires the presence of the Gfrα–3 receptor. Another provocative discovery was that Psp and to a lesser extent Ntn, but not Gdnf or Art, supported sympathetic neurons in rat SCG at P0 (Figure 5C). Finally, Art FIGURE 5.—ART is a survival factor for peripheral neurons in vitro. A, Art effectively enhanced sensory neuron numbers in DRG of wild-type rats (P2) in a dose-dependent manner. Both E coli- and CHO-cell derived mouse Art exhibited efficacy, indicating that glycosylation was not absolutely required for activity. Art 112-224 (the predominant mammalian product) was modestly more effective in stimulating neuronogenesis than Art 119-224. B, Art (20 ng/ml) did not support survival of DRG neurons of Gfrα–3 null mutant animals at P1. C, Psp and Ntn, but not Gdnf and Art, sustained SCG neurons from neonatal rats (PO). D, Addition of Art 112-224 resulted in a modest (2-fold) but significant increase in numbers of rat enteric neurons at E15. Downloaded from tpx.sagepub.com by guest on May 24, 2011 Vol. 32, No. 3, 2004 ARTEMIN INDUCES NEURAL PROLIFERATION (20 ng/ml) induced a modest (2-fold) but significant increase in neuronal numbers in rat enteric ganglia at E15 (Figure 5D); at the same concentration, Gdnf and Ntn produced 6-fold and 8-fold elevations, respectively, while Psp was ineffective. An increase in Art concentration to 100 ng/ml did not elicit any additional effect on enteric neuronal numbers. Art and Gfrα–3 Are Expressed in Contiguous Mesenchymal and Neural Domains Human Tissues: ART expression was apparent as two strong bands at ∼4.3 and ∼1.7 kb in placenta, pancreas, and prostate (Figure 6). Modest expression of both bands was also observed in adult pituitary gland (not shown), trachea (not shown), testis, ovary, small intestine, and colon, while modest expression of only the 1.7 kb band was observed in kidney (Figure 6). All other tissues and peripheral blood leukocytes had minimal to no ART mRNA (not shown). This pattern is consistent with that reported previously for ART expression in adult humans (Baloh et al., 1998). Mouse Tissues: Strong Art bands occurred at ∼1.4 and ∼1.0 kb in testis and uterus, while weaker signals were observed in thyroid, prostate, and epididymis (data not shown). 283 A similar pattern was apparent using in situ hybridization and ribonuclease protection assays for Art (data not shown). Testicular labeling resulted from signal in spermatogenic cells of the seminiferous tubules, while diffuse labeling was noted in the mucosal linings of the ureter, urinary bladder, and uterus as well as in the thyroid epithelium. In addition, multiple foci in the gastrointestinal lamina propria, renal medulla and respiratory mucosa (bronchial and tracheal linings) were labeled. The nature of the cells expressing Art at these latter sites (e.g., epithelial vs. neuronal projections) could not be identified with certainty. In developing mice, in situ hybridization for Art revealed that expression cycled both spatially and temporally during middle and late gestation (Figure 7A). At E10.5, Art signal was prominent in the primitive mesenchyme dorsal to the aorta and was apparent to a lesser extent in the adjacent lateral plate mesoderm. Beginning at E11.5, Art message began to coalesce in the differentiating mesenchyme making up the vertebral bodies. At E12.5 and E13.5, Art was expressed strongly in the cartilaginous models of various bones (chiefly the vertebrae and skull), embryonic mesentery, and the walls of the midgut loop. The skeletal labeling was most intense in the vertebral bodies surrounding the notochord and was also FIGURE 6.—ART expression in human tissues. Human multiple tissue Northern blot (left side) and human multiple tissue Northern blot II (right side) after hybridization with a 32 P-labeled DNA probe consisting of nucleotides 484 to 672 of the human ART open reading frame. Downloaded from tpx.sagepub.com by guest on May 24, 2011 284 BOLON ET AL. TOXICOLOGIC PATHOLOGY FIGURE 7.—Art expression during gestation occurs chiefly in autonomic neural and skeletal anlagen. A, In developing mice, Art expression followed both spatial and temporal cycles. At earlier stages (E10.5), Art was diffuse in dorsal and lateral mesenchyme. Expression during mid-gestation (E11.5–E13.5) was constrained to cartilage models for various vertebral anlagen (denoted by yellow box). Near term (E18.5), Art signal in the skeleton was weak and localized to cartilage. B, Strong labeling also was observed during middle (E13.5) and late (E18.5) gestation in the mesentery and outer colonic wall near elements of the autonomic nervous system, in the mucosal layer of major bronchi, and (in adults) in the seminiferous epithelium. Abbreviations: B = bronchus, I = intestine, J = joint, L = liver, N = notochord, P = pancreas, S = seminiferous tubule. Isotopic in situ hybridization for [33 P]Art (darkfield panels), with HE counterstains (brightfield panels). Bars = 100 µm. Downloaded from tpx.sagepub.com by guest on May 24, 2011 Vol. 32, No. 3, 2004 ARTEMIN INDUCES NEURAL PROLIFERATION considerable in the primitive mesenchymal cells of the periosteal anlagen; message was most prominent in primordia of the cartilaginous components (Figures 7A, 8A). A weak, diffuse signal was noted at this time in the adjacent musculature. By E18.5, Art signal in the vertebra was weak and localized only in the cartilaginous end plates (Figure 7A). In addition, strong labeling at E18.5 was observed in the mesentery anchoring most intestinal loops and within the outer wall of the colon near elements of the autonomic nervous system (Figure 7B); the location of these signals corresponded to the positions of sympathetic and myenteric ganglia, respectively. Additional sites of Art expression were observed at several times, including the epithelial layer of the dental lamina (data not shown) and in connective tissue surrounding the vas deferens at E15.5 and the mucosal layer of large bronchi at E18.5 (Figure 7B). The intensity of testicular labeling differed between tubules, apparently in accordance with the various stages of spermatogenesis (Figure 7B). Expression of Gfrα–3 was examined in tissues of developing (Figure 8) and adult mice by enzyme histochemistry (for Gfrα–3/lacZ in Gfrα–3 gene targeted mice) and in situ hybridization (wild-type animals). Gfra–3 was strongly expressed at E10.5, E12.5, and E13.5 in the DRG and sympathetic chain (para-aortic) ganglia, the spinal nerve roots, and somatic nerves. Significantly, Ret but not Gfra-1 and Gfra-2 were expressed in sympathetic chain at this same stage (data not shown), indicating that Gfrα–3 plays a major role in regulating differentiation of these structures. Signal also was observed in the mesentery and salivary gland in association with perivascular cells, likely representing components of the sympathetic nervous system. In adult tissues (data not shown), Gfrα–3 signal was observed in the DRG but was not apparent in other peripheral ganglia (sympathetic or parasympathetic, including adrenal medulla) or in peripheral nerves (sensorimotor or autonomic). A generally comparable pattern but with more widespread autonomic neural labeling was observed in embryos in which Gfrα–3 had undergone targeted replacement by Gfrα–3/lacZ. Whole mount staining for lacZ at E9.5 revealed diffuse staining of the dorsal mesenchyme surrounding the spinal cord primordium (data not shown). By E10.5, the diffuse staining was coalescing into focal condensations representing the DRG and their ventral roots, chiefly in the thoracolumbar region (Figure 8B). At E12.5 (Figure 8), Gfrα–3 expression was localized to the peripheral nervous system and occurred in both the somatic (DRG, limb and trunk nerves) and autonomic components (adrenal medulla, end-organ autonomic ganglia, SCG, sympathetic chain ganglia, trigeminal ganglia). Serial cryosections prepared from whole mount-stained E12.5 embryos also revealed diffuse staining in large fields of lateral plate mesoderm (data not shown). Gfrα–3 Null Mutant Mice Exhibit Ptosis Pups were delivered in the expected mendelian ratio. Among 152 newborns analyzed, 42 (27.6%) were homozygous mutants, 77 (50.7%) were heterozygous, and 33 (21.7%) were wildtype mice. Both heterozygous (Gfrα–3+/− ) and homozygous (Gfrα–3−/− ) mutant offspring appeared normal at birth and were indistinguishable from their wild-type littermates (Gfrα–3+/+ ) by visual inspection. The homozygous 285 mutants were all viable and fertile. Up to 12 months of age, substantial behavioral and anatomic abnormalities have not been observed. As previously reported (Nishino et al., 1999), mild ptosis was observed in 2 of 4 adult null mutants but was lacking in wild-type (n = 5) and Gfrα–3+/− (n = 7) animals. Art Expression Throughout Development Yields Systemic Expansion of Autonomic Neurons Young adult mice of both sexes that expressed enhanced levels of Art in all tissues (β-actin Art construct) or as a hepatic secretory protein (ApoE Art construct) exhibited substantial expansion of neural populations at multiple sites in the peripheral autonomic nervous system. Comparable lesions were produced by both Art-bearing transgenes. The most prominent finding was fusion and dysgenesis of cells in the adrenal medulla and an adjacent paraganglion (Figure 9). Typically, this change presented as a large, firm mass that effaced the adrenal gland. In milder lesions (Figure 9B), the caudal pole of the adrenal gland was broached, and the distribution of the adrenal cells into distinct cortical and medullary regions was disrupted. Paraganglionic neurons were mingled with chromaffin cells in the medulla, extended on occasion into the overlying cortex, and clustered in the periadrenal fat. In more extensive cases (Figure 9C), the adrenal medulla and much of the cortex was replaced by proliferating neurons enveloped in dense sheets of neuronal processes, and the paraganglia were enlarged. Adrenal lesions were associated with modest over-expression of Art (Figure 10E) and marked over-expression of Gfrα–3 (Figure 10F). Another major change was marked hyperplasia of autonomic nerves and ganglia in the pelvic connective tissue (Figure 9G) and the wall of the urinary bladder (Figure 10J); these neural elements were almost invisible in control mice (Figure 10G). This pelvic ANS locus normally is comprised of a large parasympathetic domain and a much smaller sympathetic fraction (Langworthy, 1965). Again, neuronal expansion at these sites was associated with profound overexpression of Gfrα–3 (Figure 10L), with a more modest increase in Art (Figure 10K). The increased neuronal numbers in both the adrenal medulla and the pelvic ganglia represented augmented populations of intermingled parasympathetic (ChATpositive) and sympathetic (TH-positive) cells (Figure 9), although the extent of the increase was much greater for THlabeled cells. Despite the extensive expansion of the adrenal medullae and pelvic ganglia, proliferating cells were rare. Finally, the size and numbers of myenteric ganglia and their constituent parasympathetic neurons were increased in the colons of many expressors (Figure 9I). Lesions were not observed in other peripheral autonomic ganglia or autonomic or somatic nerves, nor were they apparent in the central nervous system. Ganglionic expansion in β-actin Art-transgenic mice was apparent as early as E12.5 (SCG) but become prominent by E14.5 at other sites in the peripheral autonomic nervous system (Figure 11). Aged mice in which Art was overexpressed all developed adrenal medullary tumors (complex pheochromocytomas or neuroblastomas; Figure 12A) and hyperplasia of autonomic ganglia (Figure 12B). Sensory and motor elements of the PNS (e.g., DRG, nerve trunks and branches) of both near-term conceptuses and adult transgenic mice Downloaded from tpx.sagepub.com by guest on May 24, 2011 286 BOLON ET AL. Downloaded from tpx.sagepub.com by guest on May 24, 2011 TOXICOLOGIC PATHOLOGY Vol. 32, No. 3, 2004 ARTEMIN INDUCES NEURAL PROLIFERATION 287 FIGURE 9.—Lifelong overexpression of Art leads to widespread neuroproliferation in the peripheral autonomic nervous system. Young adult Art-transgenic mice of both sexes that expressed transgenic ART in all tissues (β-actin ART construct) or as a hepatic secretory protein (ApoE ART construct) had enlarged enteric, parasympathetic, and sympathetic ganglia. The most prominent finding was fusion and dysgenesis of sympathetic elements in the adrenal medulla and an adjacent paraganglion. In milder lesions (B), adrenal organization into distinct cortical and medullary regions was disrupted by the incursion of paraganglionic neurons. In more extensive cases (C), the adrenal medulla and much of the cortex was replaced by numerous neurons and neuronal processes. Another major change was marked hyperplasia of sympathetic (D; anti-tyrosine hydroxylase) cells and modest enhancement of parasympathetic neurons (E; anti-choline acetyltransferase) in the pelvic ganglion (a mixed ganglion with predominantly parasympathetic function) adjacent to the urinary bladder (G). Finally, the size and numbers of myenteric ganglia and autonomic nerves were increased in the colons and mesentery of ART expressers (I; anti-PGP 9.5). Comparable changes were induced regardless of whether Art over-expression was driven by the β-actin (B, D, E, I) or apoE (C, G) promoters. Controls are in A, F (normal ganglion denoted by arrowhead), and H (anti-PGP 9.5). Abbreviations: pr = prostate, ub = urinary bladder. Immunostained sections (D, E, H, I) were counterstained with hematoxylin; other sections were stained with HE. Bars = 250 µm (A—C, F, G) or 100 µm (D, E, H, I). apparently were unaffected by sustained Art overexpression (data not shown). Excess Art During Adulthood Induces Adrenal Hyperplasia or Metaplasia Following radiation-induced bone marrow ablation, Art was highly expressed in bone marrow and spleen of mice reconstituted with Art-transfected hematopoietic stem cells but not in those given stem cells bearing a retroviral vector bearing a nonsense gene (data not shown). In contrast to the system-wide autonomic expansion induced by Art overexpression throughout development, lesions were limited to the adrenal gland when Art was added during early adulthood. The changes—focal to multifocal hyperplasia FIGURE 8.—Gfrα–3 expression during gestation occurs in peripheral neural tissues and is located at sites contiguous to Art domains. A, In situ expression of Art and Gfra–3 mRNA in wild type embryos occurred in contiguous domains, with Art confined to tissues of mesenchymal origin and Gfrα–3 to neural elements. Transverse sections through the cervical and lumbar vertebral regions are represented in the left and right panels, respectively. B, Using lacZ enzyme histochemistry to detect Gfrα–3/lacZ in null mutant embryos, Gfrα–3 was localized to autonomic (adrenal medulla [A], atrioventricular node [N], superior cervical ganglion [SCG], sympathetic chain ganglia [SC] and nerve trunk [SCn]), cranial nerve (trigeminal [T]) and sensory (dorsal root ganglia [DRG]) ganglia as well as autonomic, somatic [P], and spinal (arrowheads) nerves. Embryos were all E12.5 except for the whole mount panel, which is E10.5. Abbreviations: F = fore limb, Sp = spinal cord. Bars = 200 µm. Downloaded from tpx.sagepub.com by guest on May 24, 2011 288 BOLON ET AL. TOXICOLOGIC PATHOLOGY FIGURE 10.—Neural expansion resulting from Art overexpression was associated with enhanced local expression of Gfrα–3. Relative to controls (first and third colomns), multifocal hyperplasia (outlined regions in HE-stained panels [top row]) of adrenal cells (second column) or autonomic ganglia in the urinary bladder wall (fourth column) was another pronounced finding in young adult Art-transgenic mice. The foci exhibited a neuronal phenotype and were associated with increased expression of Art and Gfrα–3 in contiguous domains. The Art transgene was driven by the β-actin promoter. Isotopic in situ hybridization for [33 P]Art or [33 P]Gfrα–3 (darkfield panels), with HE counterstains (brightfield panels). The magnification bar (A) applies to all panels. and/or neuronal metaplasia—occurred in animals supplemented with Art by administration of either recombinant Art or an Art-bearing retroviral vector (Figure 13). The conical hyperplastic foci extended from the corticomedullary junction into the inner cortex. Both lesions had not been observed previously in hundreds of age-matched (young adult) control mice in our B6D2F1/CrlBR colony, nor were they found in control mice given vehicle or retroviral vector alone. The cells in hyperplastic regions had morphologic features consistent with those of adrenocortical cells but were TH-positive (Figure 13). One day after the final subcutaneous Art dose, mitotic figures and apoptotic cells were scattered throughout the hyperplastic foci, thereby suggesting that these lesions could regress in the absence of sustained Art exposure. In contrast, metaplastic foci consisted of TH-positive neurons and neuronal processes (Figure 13) but contained no mitotic figures or apoptotic cells. The neuronal processes extended into the outer medulla. Recombinant Art Does Not Relieve Neuropathic Pain Administration of Art twice daily for 5 days by either SC bolus injection or intrathecal infusion did not alleviate mechanical allodynia in rats with ligated spinal nerve roots at any time-point tested (Figure 14). This result was not a consequence of Art degradation during storage because the molecule retained its activity toward DRG neurons during subsequent in vitro assays (data not shown). Rather, it likely reflects an absence of Art involvement in regeneration of somatic nerves in adult animals (Hoke et al., 2000). DISCUSSION ART (Baloh et al., 1998; Masure et al., 1999), the principal ligand of GFRα–3, supports neuron survival in many autonomic and sensory ganglia in the PNS as well as certain populations in the CNS (Rosenblad et al., 2000). However, ART and GFRα–3 are minimally expressed in the CNS in vivo (Baloh et al., 1998; Masure et al., 1999), suggesting that this signaling pathway acts primarily to influence PNS development under normal physiological conditions. Our data indicate that the capacity of supplemental Art to support peripheral nerve regeneration in vivo was negligible (Figure 14). However, our experiments yielded new evidence to clarify discrepancies between these initial reports while revealing important new aspects of ART biology—in particular that ART regulates neural cell populations in the adrenal medulla as well as enteric and parasympathetic ganglia. First, our data are relevant to resolving disparities in the ART literature because our nucleic and amino acid sequences are identical to the registered entries for ART (Baloh et al., 1998; Masure et al., 1999; Rosenblad et al., 2000). One laboratory described a single 113 aa transcript (Baloh et al., 1998); another demonstrated a series of 5 splice variants, of which 2 pro-peptides can be processed to yield a single mature 113 aa peptide (Masure et al., 1999); while a third reported three splice variants of 113, 116, and 140 aa (Rosenblad et al., 2000). Our data support the presence of a single ART gene with three mature peptides (104, 106, and 113 aa) resulting from alternative cleavage of a single pro-peptide (Figure 3). Only the 104 and 113 aa forms are produced in mammalian cells (Figure 3), but all three support Downloaded from tpx.sagepub.com by guest on May 24, 2011 Vol. 32, No. 3, 2004 ARTEMIN INDUCES NEURAL PROLIFERATION 289 FIGURE 11.—Neural lesions associated with Art overexpression are initiated during gestation. Mouse conceptuses bearing an Art transgene (driven here by the β-actin promoter) developed substantial neuroproliferative changes at multiple sites in the peripheral autonomic neural system during organogenesis. [Art expression driven by the apoE promoter yielded comparable changes.] The most prominent lesions were dysplasia (A, B) of the adrenal medulla, often with fusion (B) to an adjacent sympathetic paraganglion (*); (E, G) hyperplasia of parasympathetic and sympathetic elements in pelvic ganglia and the urinary bladder wall (arrowheads); and (I) massive enhancement of the superior cervical ganglion (arrowheads). All panels are at E17.5 except A (E14.5), and are stained with HE except for F and G (anti-tyrosine hydroxylase). Controls (E17.5) are shown in C, D, F, H. Abbreviations: es = esophagus, kd = kidney, Sp = spinal cord, thy = thymus, tr = trachea, ub = urinary bladder. Bars = 250 µm, except for H, I at 500 µm. FIGURE 12.—Neural lesions associated with Art overexpression progressively expand over time. The most prominent lesions were pheochromocytoma (A [arrowheads in inset]) of the adrenal medulla and expansion of the pelvic parasympathetic ganglia (B [pg and arrowhead]). Female Art-transgenic mouse (β-actin promoter), 75 weeks of age. Abbreviations: c = cervix, ub = urinary bladder. Stains: Panel A, HE; Panel B, anti-NFP with hematoxylin counterstain. Bars = 250 µm. Downloaded from tpx.sagepub.com by guest on May 24, 2011 290 BOLON ET AL. TOXICOLOGIC PATHOLOGY FIGURE 13.—Art induces adrenal medullary hyperplasia and neuronal metaplasia in adult mice. In contrast to the system-wide autonomic neuroproliferation induced by Art overexpression throughout development (Figures 7, 8), lesions produced by Art exposure during adulthood were limited to the adrenal corticomedullary junction and consisted of hyperplasia (A, B) and neuronal metaplasia (C, D). Hyperplasia occurred chiefly when Art was administered by subcutaneous injection (5 mg/kg/day for 14 consecutive days), while metaplasia developed in mice made transgenic for Art using a retroviral vector. Cells in the hyperplastic foci had morphologic features consistent with those of cortical cells but expressed tyrosine hydroxylase (B; anti-TH); apoptotic cells and mitotic figures were frequent. In contrast, the metaplastic foci were comprised of small neurons (∗ ) in association with disordered bundles of neurites (C, arrowhead). The neurons as well as isolated medullary cells (arrowheads) expressed the neuronal marker PGP 9.5 (D; anti-PGP 9.5). Immunostained sections (B, D) were counterstained with hematoxylin; other sections were stained with HE. Bars = 200 or 50 (inset only) µm. FIGURE 14.—Art therapy does not alleviate neuropathic pain (allodynia). Surgical ligation of the lumbar spinal nerve roots (L5 and L6) substantially decreased the paw withdrawal threshold (PWT) to graded tactile stimuli ranging from 4.0 to 148.1 mN (0.41 to 159). Administration of Art 119–224 twice daily for 5 days by either IP injection (5 mg/kg) or intrathecal (IT) administration (10 µg unit dose) did not reduce or reverse mechanical allodynia. Abbreviation: Sx = surgery. Downloaded from tpx.sagepub.com by guest on May 24, 2011 Vol. 32, No. 3, 2004 ARTEMIN INDUCES NEURAL PROLIFERATION the survival of mammalian neurons (Figure 5A). It is unclear at present which molecule is preferred in vivo or whether the forms specify different activities at diverse sites. A second point addressed by our studies was the interaction of ART with GFRαs. The high affinity ART receptor is clearly GFRα–3, but neither a competing laboratory (Masure et al., 1999) nor our data (Figures 4, 5) corroborate a proposed low affinity ART-GFRα–1 pairing (Baloh et al., 1998; Milbrandt et al., 1998). However, it cannot be ruled out that supraphysiological ART concentrations might mediate productive signaling through other GFRα receptors, a possibility supported by the upregulation of ART in rat striatum ipsilateral to a chemical lesion (Zhou et al., 2000) and the capacity of ART gene therapy to ameliorate chemical lesions to mesencephalic dopaminergic neurons (Rosenblad et al., 2000) despite very low levels (Stover et al., 2000) or absence (Naveilhan et al., 1998; Widenfalk et al., 1998) of GFRα–3 expression at this site. Further work is needed to resolve this question. Development of many PNS ganglia is regulated by GDNF and NTN (Durbec et al., 1996; Golden et al., 1998; Heuckeroth et al., 1999; Enomoto et al., 2000). Our present expression and functional data indicates that ART also plays a critical role in differentiation and maintenance of these structures. The primary populations regulated by ART/GFRα–3 signaling are sensory and sympathetic neurons (Baloh et al., 1998), including adrenal medulla (Masure et al., 1999). Our data confirm that Gfrα–3 is strongly expressed in these locations (Figure 8B); in fact, in mouse sympathetic chain anlagen, Gfrα–3 and Ret but not Gfrα–1 or Gfrα–2 were present (Figure 8A), suggesting that ART is the principal GDNF-like neurotrophic factor acting at this site. Our findings suggest that Art has no effect on DRG neurons during gestation but instead acts after birth, thus confirming that DRG exhibit developmental stage-specific sensitivity for GDNF-like ligands (Baudet et al., 2000). A similar scheme may be active in the SCG in that Psp and Ntn, but not Gdnf and Art, supports SCG neuron survival at P0 (Figure 5C), while a prior report reveals that SCG are sensitive to all three ligands at P1 (Baloh et al., 1998). In like manner, the ability of GDNF to support enteric neuron survival varies over time due to changes in expression of GFRα–1 (Worley et al., 2000), while dynamic spatial and temporal cycling of GDNF, NTN and their receptors coordinate cutaneous sensory innervation (Fundin et al., 1999). Further work is required to test whether or not this premise represents a general paradigm for GDNF family members for other ANS neurons. Finally, our data provide compelling evidence that ART regulates development of enteric and some parasympathetic neurons. Expression of Ret is necessary for normal development of neural crest cells of the sympathoenteric lineage, which differentiate into parasympathetic cells of the enteric nervous system as well as sympathetic cells of the SCG (Durbec et al., 1996; Watanabe et al., 1997). Addition of Art at physiological concentrations induced a modest but significant increase in neuron numbers in rat enteric ganglia cultures (Figure 5D). Furthermore, Art was strongly expressed in the outer colonic wall (Figure 7B), the location of the myenteric ganglia, while Art-transgenic mice had more and larger myenteric ganglia and pelvic (chiefly parasympathetic) ganglia (Figure 8). The response to Art in these structures was mild relative to effects demonstrated in sympathetic ganglia 291 and adrenal medulla. Nevertheless, ART appears to be another in the growing number of ligands that regulates enteric neurogenesis. Significantly, several facets of our investigation raised the prospect that ART/GFRα–3 signaling has additional roles in bone and/or bone marrow biology. The pattern and timing of Art (Figure 7) and Gfrα–3 (Figure 8) expression cycled both spatially and temporally during organogenesis. Initially, Art and Gfrα–3 were distributed diffusely in dorsal mesenchyme, after which their expression was restricted to distinct but contiguous regions of the nervous system (Art and Gfrα–3) and skeleton (Art). These boundaries suggested that ARTmediated neurotrophic activity depends on paracrine interactions between adjacent (neuro)ectodermal or endodermal and mesenchymal derivatives, a paradigm already demonstrated for GDNF (Wartiovaara et al., 1998; Golden et al., 1999; Saarma and Sariola, 1999). Intriguingly, however, we first cloned Art from severely osteoporotic bones of osteoprotegerin (Opg) knockout mice (Bucay et al., 1998). GFRαs have been postulated to regulate hematopoietic differentiation (Gattei et al., 1997). Taken together, these findings support the speculation that ART might participate in hematopoietic and/or skeletal development. This possibility is made more plausible by recent reports that other TGFβ superfamily members that regulate skeletal development (bone morphogenetic proteins [BMP]) also contribute to sympathoadrenal differentiation (Varley and Maxwell, 1996; Schneider et al., 1999; McPherson et al., 2000; Sasai, 2001), and that neurotrophic factors may exert influence on bone marrow (Labouyrie et al., 1999). Finally, our findings strongly suggest that neuroproliferative changes elicited by ART therapy will overshadow any potential neuroprotective benefits. In humans, activating mutations in RET (Takahashi et al., 1999; Edström et al., 2000) have been demonstrated as genetic events leading to both sporadic pheochromocytomas and multiple endocrine neoplasia (MEN, a syndrome in which pheochromocytoma is a prominent feature) but not paragangliomas (Edström et al., 2000). Constitutive activation of RET under the control of the dopamine-β-hydroxylase promoter (which specifies expression in sympathetic cells) results in neuroglial hyperplasia in adrenal medullae and sympathetic ganglia in transgenic mice (Gestblom et al., 1999). Similarly, mice engineered to express murine Ret containing the specific MEN2B mutation develop pheochromocytomas and sympathoadrenal hyperplasia (Smith-Hicks et al., 2000). Neither GDNF/GFRα–1 (Myers et al., 1999; Edström et al., 2000) nor NTN/GFRα–2 (Edström et al., 2000) are found in human pheochromocytomas, nor does GDNF serve as a growth factor for normal or neoplastic chromaffin cells (Powers et al., 1998). While the neoplastic effects in thyroid glands in MEN2 have been imputed to GFRα–4 rather than GFRα–3 (Lindahl et al., 2000, 2001), our data showing that overexpression of Art in transgenic mice induced multifocal neuronal expansion in adrenal medulla (Figures 9, 11, 12) introduces the possibility that excessive ART production provides a means of initiating neoplasia in chromaffin cells. The mature adrenal medulla retains a population of undifferentiated chromaffin cells (Chen-Pan et al., 2002), which possess neurogenic capabilities (reviewed in (Martinez and Mog, 2001)); a reasonable hypothesis is that the neuronal metaplasia in Downloaded from tpx.sagepub.com by guest on May 24, 2011 292 BOLON ET AL. adrenal glands of adult Art-transgenic mice (Figure 13) resulted from the Art responsiveness of this stem cell population. Furthermore, ART acts in vitro to promote the growth of SH-SY5Y cells, a human neuroblastoma cell line originating from adrenal sympathetic cells (Baloh et al., 1998; Masure et al., 1999). Considered together, these data lend credence to the hypothesis that chronic ART overexpression leading to persistent Ret activation might be important steps in the pathogenesis of sympathoadrenal neoplasia. It is noteworthy that the location of the human ART gene— chromosome 1p31–33 (Baloh et al., 1998; Masure et al., 1999)—is in or immediately adjacent to the locus of the most common early gene deletion in human pheochromocytoma and paragangliomas (Dannenberg et al., 2000; Edström et al., 2000). The significance of this association is unknown as these tumors are postulated to result from the loss of an unknown tumor suppressor gene (Dannenberg et al., 2000; Edström et al., 2000) while ART presumably acts as a growth factor. 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