The FASEB Journal express article 10.1096/fj.03-0291fje. Published online October 2, 2003. Sonic hedgehog is a neuromodulator in the adult subthalamic nucleus Erwan Bezard,*,�,** Jerome Baufreton,�,** Geraint Owens,� Alan R. Crossman,�,§ Hank Dudek,║ Anne Taupignon,� and Jonathan M. Brotchie#

*Basal Gang, and �CNRS UMR 5543, Université Victor Segalen, 146 rue Léo Saignat, 33076 Bordeaux Cedex, France; �Manchester Movement Disorder Laboratory, Division of Neuroscience, School of Biological Sciences, University of Manchester, 1.124 Stopford Building, Manchester, M13 9 PT, United Kingdom; §Motac Neuroscience Ltd., Williams House, Manchester Science Park, Lloyd Street North, Manchester, M15 6SE, United Kingdom; ║Curis, Inc., 61 Moulton Street, Cambridge, Massachusetts; and #Toronto Western Research Institute, MC 11-419, Toronto Western Hospital, 399 Bathurst Street, Toronto, Ontario M5T 2S8, Canada. **These two authors have contributed equally.

Corresponding author: Erwan Bezard, Basal Gang, and CNRS UMR 5543, Université Victor Segalen, 146 rue Léo Saignat, 33076 Bordeaux Cedex, France. E-mail: erwan.bezard@umr5543.u-bordeaux2.fr

ABSTRACT

It is well established that members of the hedgehog family are involved in tissue patterning during development. We herein show that sonic hedgehog signaling molecules are differentially regulated by dopamine depletion in the basal ganglia of adult animals and specifically that sonic hedgehog levels are reduced in an animal model of Parkinson�s disease. In addition, we show that sonic hedgehog protein inhibits electrical activity in the subthalamic nucleus, a key element of basal ganglia, within minutes of application. As the subthalamic nucleus is overactive in parkinsonism, we suggest that enhancement of sonic hedgehog signaling in the subthalamic nucleus may be of therapeutic value in Parkinson�s disease.

Key words: patched • Parkinson�s disease • basal ganglia • rat • macaque

t is well established that members of the hedgehog family are involved in tissue patterning during development (1). Members of the hedgehog (hh) family encode a class of secreted proteins that act as local intercellular signals. Sonic hedgehog (shh), together with the genes

encoding its signaling proteins�including patched (ptc), the purported receptor mediating at least some of the actions of shh (2) and smoothened, a downstream regulatory molecule (3)�have been found to be expressed in functionally related regions of the adult rat brain (4). The combination of the neuroprotective properties of shh (5); its ability to induce neuron proliferation and differentiation, including midbrain dopaminergic neurons (6, 7); and the persistence of shh expression in various tissues into adulthood (e.g., 8, 9), has aroused interest in the functional properties of shh and its signaling components in adults. However, there is no functional evidence to indicate what role(s) shh may have in the brain of adult animals.

I

Interestingly, globus pallidus (GP) neurons, which project onto subthalamic nucleus (STN) neurons, appear to express shh, whereas STN neurons appear to express its receptor, ptc (4). The GP-STN pathway is a key component of the circuitry involved in the symptoms of Parkinson�s disease (10). Hypoactivity of pallidal neurons is thought to be responsible for pathological STN hyperactivity (11). This over activity of STN is a critical component of the neural mechanisms underlying the generation of parkinsonian symptoms (12). In fact surgical procedures to directly reduce the activity of the STN have dramatic anti-parkinsonian benefit (for review, see 13), and to date no pharmacological therapy has been described that can mimic the effects of such surgery.

Considering that i) shh is a secreted protein whose mRNA is present in presynaptic neurons (4), i.e., in GP neurons; ii) shh protein is anterogradely transported in axons (14-16); and iii) the expression of ptc appears to occur in postsynaptic neurons (4), i.e., in STN neurons, we hypothesized that shh conveys a message from presynaptic neurons to postsynaptic neurons in the GP-STN pathway. Also, we wondered how shh and ptc mRNA are modulated in the 6-hydroxydopamine (6-OHDA)-treated rat model of Parkinson�s disease.

MATERIALS AND METHODS

Animals

Experiments were performed on i) 18�22 day-old inbred Wistar rats (electrophysiology) and ii) male Sprague Dawley rats (250�300 g, Charles Rivers, UK; DAT binding, in situ hybridization) Animals were housed in a temperature-controlled room under a 12 h light/dark cycle (08.00�20.00) with free access to food and water. Experiments were performed in accordance with European Communities Council Directive of 24 November 1986 (86/609/EEC) for care of laboratory animals. All efforts were made to minimize animal suffering and to use the minimum number of animals necessary to perform statistically valid analysis. In situ hybridization studies were also conducted on tissue prepared from an adult 3 kg female macaque monkey (Macaca fascicularis, SAH, Beijing, People�s Republic of China), which had been used in previous experiments (17, 18).

Electrophysiological recording of STN neurons

Experiments were performed on subthalamic neurons in 400-µm-thick coronal slices of rat brain as described previously (19, 20). Briefly, after killing by cervical dislocation, the rats were decapitated, brains were rapidly removed, and a block of cerebral matter containing the STN was isolated in an ice-cold sucrose-enriched solution. After a 2 h recovery period in a Krebs� solution containing (in mM): 124 NaCl, 26 NaHCO3, 3.6 KCl, 1.3 MgCl2, 2.4 CaCl2, 1.25 HEPES, and 10 glucose (pH 7.4), bubbled with 95% O2 and 5% CO2; one slice was transferred to an immersion-type recording chamber (21) and continuously superfused (3.5 ml/min) with the oxygenated Krebs� solution. The slice was examined under a dissecting microscope; STN was readily identified as ovoid gray matter immediately dorsal to the cerebral peduncle. Cell-attached experiments were performed. Tight seals were obtained by using a pipette containing Krebs�solution. The patch was held at bath potential in the voltage clamp mode. Action potentials were therefore seen as action currents (22). On-cell activity was continuously recorded on a videotape. ShhN [amino acids 1�201 of full-length shh, this represents the N terminus of

shh proteins, which has previously been shown to contain the biologically active domain of shh and be more potent in mediating shh actions than the full length shh itself (2)] was diluted in the oxygenated Krebs� solution and delivered by means of a multi-barrel gravity-feed system (HSSE-2, ALA Scientific Instruments Inc., Sega Electronique, France) composed of two capillaries positioned just above the patch pipette. Time taken by a drug to consistently reach a neuron under recording has been evaluated to 2 s (19). All our records came from naïve neurons. Once a slice had been perfused with shhN, it was discarded and experiments were performed on another slice.

Data analysis

Recordings were analyzed by using pClamp 6.01 software (Axon Instruments, Foster City, CA), Origin 5.0 (Microcal, Northampton, MA), and Instat (GraphPad Software, Inc., San Diego, CA). Action currents were detected by the threshold method using the Fetchex subroutine of pClamp. The cumulative number of action potentials was registered every 30 s. It was transformed off-line in mean action potential frequency over the 30 s bins. The mean firing frequency in control was then calculated and used to normalize all values in a record. In the graphs, values are shown as mean (thick trace) and standard error (gray envelop).

Unilateral lesion of the medial forebrain bundle

The procedure for producing this unilateral animal model of Parkinson�s disease has been described in detail on many occasions (e.g., 23, 24). Thirty minutes prior to surgery, animals were injected with pargyline (5 mg/kg, Sigma) and desipramine (25 mg/kg; 1 ml/kg i.p. in sterile 0.9% w/v sodium chloride; Sigma). Under halothane (Fluorothane, Zeneca Ltd., Macclesfield, UK) anesthesia, rats were positioned in a stereotaxic frame (Kopf, Tujunga, CA). Each animal received a unilateral injection of 2.5µl 6-OHDA.Br (Sigma, 5 mg/ml in sterile water with 0.1% ascorbic acid) into the right medial forebrain bundle at coordinates 2.8 mm rostral to Bregma, 2 mm lateral to the midline, and 9 mm below the skull (25). On Day 21, rats were killed by cervical dislocation. The brains were removed, rapidly frozen in isopentane cooled to � 45°C, and stored at �70°C until further analysis. Coronal brain sections were cut at �19°C (20 µm thick) by using a cryostat (OTF, Bright, UK) and were thaw-mounted onto gelatin/chrome-alum-coated slides.

Dopamine transporter (DAT) binding

The radiolabeling of the selective DAT ligand, [125I]-(E)-N-(3-iodoprop-2-enyl)-2β-carboxymethyl-3β-(4'-methylphenyl)-nortropane (PE2I), was performed from the stannyl precursor according to a method described previously in order to identify the dopaminergic nerve endings (26). After purification, [125I]PE2I was obtained in a no-carrier-added form with a specific activity of 2000Ci/mmol. [125I]PE2I was kept in ethanol at �20°C and is stable for 1 month under these storage conditions (27). Sections were incubated for 90 min at 25°C with 100 pM [125I]PE2I in pH 7.4 phosphate buffer (NaH2PO4 10.14 mM, NaCl 137 mM, KCl 2.7 mM, KH2PO4 1.76 mM) as described previously (17, 28). Adjacent sections were incubated in the presence of 100 µM cocaine (Sigma, St Louis, MO) to define non-specific binding. Following incubation, sections were washed twice for 20 min in phosphate buffer at 4°C and then rinsed for 1 s in distilled water at 4°C. After drying at room temperature, sections were exposed to β

radiation-sensitive film (Hyperfilm ßmax, Amersham, UK), together with calibrated [125I]-microscales (Amersham) in X-ray cassettes, for 3 days.

Preparation of non-human primate tissue

The macaque animal was deeply anesthetized by sodium pentobarbital overdose (150 mg/kg, i.v.), and the brain was removed quickly after death as described previously (17, 18). The brain was bisected along the midline, and the two hemispheres were immediately frozen by immersion in isopentane (�45°C) and then stored at �70°C. Tissue was sectioned at 20 µm in a cryostat at �17°C, thaw-mounted onto gelatin-subbed slides, and stored at �70°C.

In situ hybridization with [α-33P] UTP-labeled cRNA probes

Fragments of mouse (642 bp; 29) and human (1645 bp D. Bumcrot, unpublished) shh, together with mouse (841 bp; 30) and human (1279 bp, D. Bumcrot, unpublished) ptc-1 cDNA sequence templates were subcloned into pBluescript II SK/KS plasmids (D. Bumcrot, Cambridge, MA). The plasmids were amplified into Library-efficiency DH11S competent cells (Life Technologies, Ltd., UK). Extraction and purification of the plasmids was performed by using a QIAGEN® midiprep kit (QIAGEN Ltd., Crawley, UK). To confirm that plasmid amplification had occurred, a sample of each amplified cDNA insert was sequenced based upon the dideoxy-chain termination method (31), using an ABI PRISM® BigDyeTM Terminators v.3.0 cycle sequencing kit (Applied Biosystems Ltd., Warrington, Cheshire, UK), and compared with the NCBI GenBank sequence database GenBank accession numbers X76290 (mouse Shh), NM_008957 (mouse Ptc-1), NM_000193 (human Shh), U43148 (human Ptc-1, http://www.ncbi.nlm.nih.gov/GenBank/). The riboprobes were transcribed in vitro from 1 mg of linearized plasmid by using an RNA transcription kit (Stratagene Inc., La Jolla, CA). Included in the reaction mixture were 5 ml transcription buffer (5× concentrate); 1 ml volumes of unlabeled rATP, rGTP, and rCTP (10 mM each); and 1 ml RNase inhibitor (20 U/ml, Boehringer Manheim, Germany). The volume was adjusted with DEPC-treated water to give a final reaction volume of 25 ml. A 5 ml volume of Uridine 5′-[α-33P] triphosphate ([33P]-UTP, 2 mM, 2500 Ci/mmol; Amersham Radiochemicals) was added to radiolabel the probes. T3 and T7 RNA polymerase (25 U/ml) were used to generate antisense and sense probes from each cDNA fragment template. The reaction mixture was incubated for 2 h at 37°C. To remove unincorporated nucleotides, the reaction mixture was passed through a MicroSpin� sephacryl S-200 HR resin column equilibrated in Tris-HCl/EDTA (TE) buffer (pH 7.6; 1000 g, 2 min, Amersham Pharmacia Biotech Inc., Piscataway, NJ). The riboprobes were precipitated with 70% ethanol/0.3 M sodium acetate and 10 mg yeast tRNA (10 mg/ml, Sigma-Aldrich Company Ltd., Poole, Dorset, UK) and extracted by centrifugation (10,000 g, 20 min). The labeled riboprobe pellet (washed twice with ice-cold 70% ethanol) was resuspended in 100 ml of 60% hybridization buffer (0.3 M NaCl, 50 mM Tris-HCl pH 8, 5 mM EDTA, 10% dextran sulfate, 2% Denhardts (50×), 2.5% yeast tRNA (10 mg/ml), 60% formamide, 10 mM DTT). The specific activity of the probe was determined by using a Packard Tricarb® 1500 liquid scintillation analyzer (Pangbourne, Berks, UK), and the final volume was adjusted to give 85,000 counts per minute (cpm)/ml.

Coronal sections of rat and macaque brain were removed from storage and allowed to reach room temperature. Following prefixation and dehydration procedures, shh or ptc-1 [33P] UTP-labeled riboprobe was applied to each section (three antisense sections and three sense sections per level). Following incubation for 18 h at 62°C and removal of unhybridized probes, the sections were air-dried and exposed to autoradiographic film (Hyperfilm βmax, Amersham, Arlington Heights, IL). The sections were exposed together with 14C autoradiographic microscale standards (30�862 pCi/mg, Amersham, Arlington Heights, IL) for 5 weeks. The optical densities were measured as described previously (17, 18), and the degree of signal was expressed as pCi (equivalent to 14C) per mg of tissue (according to the 14C autoradiographic microscale standard). Hybridization to sections of macaque brain tissue was analyzed qualitatively.

RESULTS

Shh protein inhibits STN electrical activity

Spontaneous electrical activity was recorded from STN in rat brain slices in the cell-attached mode by using the patch clamp technique (19, 20). The cell-attached mode was chosen to prevent possible disruption of unknown intracellular transduction pathway(s), because it provides a physiological test on an intact neuron in a slice. Spontaneous activity was examined under basal conditions for 12 min and was found to consist of regularly firing single spikes, in agreement with other on-cell studies (19, 32, 33). To reveal shhN action, two sequential applications of shhN, lasting 1 and 10 min, respectively, were made at an interval of 10 min. shhN was used at 10 nM, a dose close to the reported EC50 (2). Vehicle was also tested by using the same paradigm.

Neither vehicle applications (n=10; not shown) nor 1 min applications of shhN (n=12) ever affected firing frequency (Fig. 1a). By contrast, shhN reduced firing frequency by 25±10% in 4 of 12 neurons tested, with a delay of 3 min (Fig. 1a). ShhN was without effect in the eight other neurons (Fig. 1b). Thus shhN had a specific inhibitory action on a subset of subthalamic neurons. This is the first evidence that shhN affects electrical activity and that it acts on over a short time scale. In the mature rat brain, in vitro, shhN has therefore a neuromodulatory activity.

Decreased shh mRNA expression in the GP of 6-OHDA-treated rat

In situ hybridization was performed by using antisense and sense (not shown) 33P-labeled shh and ptc-1 mRNA probes in coronal sections of rat brain at GP and STN levels, in the 6-OHDA lesioned rat model of Parkinson�s disease. Striatal 125I-PE2I binding was reduced by over 90% on the 6-OHDA-lesioned side, compared with the unlesioned side, in all animals included in the analysis. In unilaterally lesioned animals, a significant decrease, of 45%, in shh mRNA level was observed in the GP on the lesioned side compared with the unlesioned side (P<0.001; Paired t-test, n=10 animals; Fig. 1c, e). ptc-1 mRNA expression in the STN, however, remained stable (Fig. 1d, f). Thus, in a well-validated animal model of PD, shh signaling in the GP-STN pathway appears to be reduced.

Similar distribution of shh and ptc-1 mRNA in the non-human primate basal ganglia

Antisense and sense (not shown) 33P mRNA probes were generated from human shh (1645 bp) and ptc-1 (1279 bp). Shh was expressed in the pars externalis of the globus pallidus (GPe), primate equivalent of the rodent GP (Fig. 1g), whereas ptc-1 was present in the STN (Fig. 1h). Both shh (Fig. 1g) and ptc-1 (Fig. 1h) are not expressed in high levels in the putamen and the pars internalis of the globus pallidus, the main output structure of non-human primate basal ganglia. The pattern of signal distribution using human shh and ptc-1 mRNA probes in the primate brain is thus very similar to that seen in the rat brain (Fig. 1g�h).

DISCUSSION

We have shown that the N-terminal fragment of shh protein inhibits electrical activity in the adult STN, a key element of the basal ganglia, within minutes of application, which suggests that shh is a neuromodulator in the basal ganglia. In addition, shh signaling molecules are differentially regulated by dopamine depletion in the basal ganglia of adult animals and specifically, shh levels are reduced in an animal model of Parkinson�s disease.

Spontaneous electrical activity was recorded in rat brain slices in the cell-attached mode, that is, on single intact neurons in vitro. The electrophysiological protocol was designed to reveal the time scale of shhN action in mature brain. We made two sequential applications of shhN separated by a 10-min delay. The first one lasted for 1 min, whereas the second one lasted for 10 min. The first application did not produce any change in firing frequency. Changes appeared only during the second application with a latency of 3 min. This suggested that shhN binding to its receptors for 3 min was required to induce detectable changes in firing frequency. However, it cannot be excluded that the action of shhN requires an initial priming.

STN neurons display spontaneous rhythmic single-spike activity when they are isolated from synaptic inputs by severing afferents and/or by the application of neurotransmitter-receptor antagonists (19, 32, 34). Regular single-spike firing is an intrinsic property of rat subthalamic nucleus neurons and is caused by the fine interplay of several time-, voltage-, and calcium-dependent conductances (32). Our results thus suggest that a potential target of shh in the adult brain might be membrane channels. Our choice of the cell-attached mode was made as this prevents dialysis of the cytosol by the pipette saline and thus avoids possible disruption of unknown intracellular transduction pathway(s). However, cell-attached mode recording did not allow analysis of the transduction pathway(s) of shh signaling. Unfortunately, because ptc, the shh receptor, is expressed only by STN neurons and not by GP neurons (Fig. 1d, f; 4), a direct, post-synaptic action of shhN onto STN neurons can be assumed and presynaptic modulation of afferent terminals kept in the slice is less likely.

Shh binds to the membrane protein ptc with high affinity (2). The predicted structure of ptc is a protein with 12 membrane-spanning domains and a symmetrical conformation of 6+6 trans-membrane domains (30, 35). This structure is reminiscent of the 6+6 family of transmembrane transporters (36), raising the possibility that ptc may function as a ligand-gated channel to allow the flow of ions across the membrane. However, expression of recombinant ptc in Xenopus oocytes does not induce any Na+, K+, Cl�, or Ca2+ currents, whether in the presence or in absence of shhN (2). Therefore, ptc itself is not likely to allow ions to permeate through the plasma

membrane. Ptc has been found to interact with another membrane bound protein, smoothened (smo; 1). It was further shown that i) ptc and smo are co-expressed in multiple tissues; and ii) they form a complex to which binds hh (37). Because smo shows structural similarities to members of the G-protein coupled receptors� super family (3), it has been suggested that smo is responsible for the transduction of an external hh signal to the cytoplasm and nucleus. In this model, ptc is considered as a hh-regulated suppresser of smo signal transduction (2, 3, 37). Smo is, however, absent from the basal ganglia in the adult brain (4). Indeed, whereas ptc and smo transcripts display an overlapped distribution in neural folds and early neural tube, they are co-localized in only a very limited number of adult brain areas and not in basal ganglia (4). This might reflect that a yet-unidentified hh binding protein is expressed in STN mature neurons. Alternatively, ptc, in the absence of any co-factor, may be sufficient to transduce fast, neuromodulatory hh signaling. It has been hypothesized that ptc, when expressed in the absence of smo, might have activities other than transducing the hh signal through its putative sterol-sensing domain (38, 39). We thus suggest that ptc transduces shh signal in the absence of smo or patched-2, a recently identified homologue of ptc (40), which is also not found in the adult basal ganglia (4). It must be remembered that all the elements of Shh transduction pathway are expressed only by a sub-population of subthalamic neurons, since shhN inhibited one neuron in three.

The net inhibitory effect of shhN onto STN neurons is of particular interest with regard to the role played by the STN in the basal ganglia. The GP-STN pathway is a key component of the circuitry involved in the symptoms of Parkinson�s disease (10). Hypoactivity of pallidal neurons is assumed to be responsible for the pathological STN hyperactivity (11) that is thought to be a critical component of the neural mechanisms underlying the generation of parkinsonian symptoms (12). Recent studies have also suggested that patterns of activity in STN and GP contribute critically to information processing in basal ganglia (for review, see 13, 41). Thus, normal information processing in the STN and GP is characterized by complex spatiotemporal patterns of firing, whereas in Parkinson�s disease, STN and GPe neurons display more correlated, synchronous, and rhythmic patterns of activity (for review, see 13, 41). Bevan and co-workers have elegantly demonstrated how STN activity may be dramatically affected by a single GABAergic input arising from the GPe (33, 42). Their work suggest that, in addition to its best-known effect on firing frequency (19, 41), the magnitude of the inhibitory postsynaptic potentials (IPSPs) drives the shift from synchronized to desynchronized activity (41), that is, from pathological to normalized mode of discharge.

With Shh likely released by the GPe neurons as GABA and inhibitory as well, it can be speculated that it plays a key role in the modulation of the magnitude of the IPSPs in STN neurons. How shh participates to the complex control of STN activity warrants further studies. Nevertheless, considering that i) STN is overactive in parkinsonism (11, 12); ii) shh inhibits STN neurons; and iii) the loss of dopaminergic input to the basal ganglia reduces shh expression in GP, we suggest that normalizing shh levels in the STN may prove useful in controlling the pathological STN hyperactivity seen in Parkinson�s disease. The presence of signal-using human shh and ptc-1 mRNA probes in the primate brain, with a similar distribution to that seen in the rat brain (Fig. 1g�h), adds further weight to the concept that enhancement of shh signaling might find therapeutic application in the symptomatic treatment of Parkinson�s disease.

ACKNOWLEDGMENTS

We wish to thank D. Bumcrot for providing the riboprobes. This work was supported by the Medical Research Council, BBSRC, the Parkinson's Disease Society (UK), the CNRS, and the University Victor Segalen-Bordeaux 2. J. B. received a doctoral fellowship from the Aquitaine Region; and G. Owens, from Curis Inc. H. D. is an employee of Curis Inc., which owns patents on therapeutic applications of shh-like compounds.

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Received May 1, 2003; accepted August 5, 2003.

Fig. 1

Figure 1. Shh inhibits electrical activity in a subset of subthalamic neurons (a), whereas it does not affect firing in other subthalamic neurons (b). Values obtained from four responsive neurons are averaged. Standard error is displayed by vertical bars. Firing frequency of each neuron is expressed in % of its mean firing frequency in the 12-min control period. Shh (c, e) but not ptc-1 (d, f) mRNA expression is reduced in the unilaterally 6-OHDA-lesioned rat. Shh (g) and ptc-1 (h) mRNA are expressed heterogeneously in the adult macaca fascicularis. Put: Putamen. GPi: pars internalis of the globus pallidus.