Abstract
Nicotinic acetylcholine receptors (nAChRs) represent an important modulator of striatal function both under normal conditions and in pathological states such as Parkinson's disease. Because different nAChR subtypes may have unique functions, immunoprecipitation and ligand binding studies were done to identify their subunit composition. As in the rodent, α2, α4, α6, β2, and β3 nAChR subunit immunoreactivity was identified in monkey striatum. However, distinct from the rodent, the present results also revealed the novel presence of α3 nAChR subunit-immunoreactivity in this same region, but not that for α5 and β4. Relatively high levels of α2 and α3 subunits were also identified in monkey cortex, in addition to α4 and β2. Experiments were next done to determine whether striatal subunit expression was changed with nigrostriatal damage. 1-Methyl-4-phenyl-1,2,3,6-tetrahydropyridine treatment decreased α6 and β3 subunit immunoreactivity by ∼80% in parallel with the dopamine transporter, suggesting that they are predominantly expressed on nigrostriatal dopaminergic projections. In contrast, α3, α4, and β2 subunit immunoreactivity was decreased ∼50%, whereas α2 was not changed. These data, together with those from dual immunoprecipitation and radioligand binding studies ([3H]cytisine, 125I-α-bungarotoxin, and 125I-α-conotoxin MII) suggest the following: that α6β2β3, α6α4β2β3, and α3β2* nAChR subtypes are present on dopaminergic terminals and that the α4β2 subtype is localized on both dopaminergic and nondopaminergic neurons, whereas α2β2* and α7 receptors are localized on nondopaminergic cells in monkey striatum. Overall, these results suggest that drugs targeting non-α7 nicotinic receptors may be useful in the treatment of disorders characterized by nigrostriatal dopaminergic damage, such as Parkinson's disease.
Parkinson's disease is a neurodegenerative disorder characterized by severe movement disability (Olanow, 2004; Samii et al., 2004). Although the underlying cause seems to be a loss of nigrostriatal dopaminergic neurons, other neurotransmitter systems are also affected. This includes the cholinergic system, in which declines have been observed in several cholinergic measures, including nicotinic acetylcholine receptors (nAChRs). Binding sites for 125I-epibatidine, [3H]cytisine, [3H]nicotine, and 125I-α-conotoxin MII are decreased in Parkinson's disease, with no change in 125I-α-bungarotoxin receptors (Gotti et al., 1997; Court et al., 2000; Quik et al., 2004). These data indicate that receptor subtypes expressing the α4β2 ([3H]cytisine and [3H]nicotine binding), and the α3β2 and/or α6β2 (125I-α-conotoxin MII sites) subunits are decreased in Parkinson's disease, whereas those containing α7 (125I-α-bungarotoxin) are not affected. Studies to identify the other nAChR subunits that comprise these nAChR subtypes are critical for the development of subtype-selective agents targeting the receptors deficient in this disorder. However, experiments using antibodies directed to human nAChR subunits have yielded uncertain results (Martin-Ruiz et al., 2000; Guan et al., 2002).
Because animal models represent an excellent first step, studies have been done in both rodents and monkeys to address this question. In rodents, numerous nAChR subunit mRNAs (α2–α7 and β2–β4) have been localized to the substantia nigra (Marks et al., 1992; Le Novere et al., 1996; Whiteaker et al., 2000, 2002; Champtiaux et al., 2002). Moreover, receptor binding and antibody immunoprecipitation studies indicate that these transcripts are expressed with multiple nAChR subtypes present in the striatum, including those expressing α4β2, α4β2α5, α6α2β2β3, and α6β2β3 (Whiteaker et al., 2000, 2002; Klink et al., 2001; Zoli et al., 2002; Champtiaux et al., 2003, 2002; Salminen et al., 2004). Nigrostriatal damage, produced by administration of the selective dopaminergic neurotoxins 6-hydroxydopamine or 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) results in losses of both α4* and α6* nAChR populations in rodents (Zoli et al., 2002; Champtiaux et al., 2003; Quik et al., 2003b). Moreover, these receptor losses are associated with functional deficits at both the cellular (Quik et al., 2003b) and behavioral (Le Novere et al., 1999) levels.
Studies to identify the nicotinic receptor subtypes and the effects of nigrostriatal damage and dopamine precursor treatment have also been done in nonhuman primates, which bear a close resemblance to humans at the genetic, molecular, and behavioral level. In addition, monkeys with nigrostriatal damage exhibit symptoms that resemble those in Parkinson's disease, with the motor deficits reversed by the same drug used to treat this disorder. Studies have shown that the α2–α7 and β2–β4 nAChR transcripts are present in monkey substantia nigra (Han et al., 2000; Quik et al., 2000a,b) and that binding sites for 125I-epibatidine, [3H]cytisine, 125I-A85380, 125I-α-conotoxin MII, and 125I-α-bungarotoxin are expressed in the striatum and substantia nigra (Quik et al., 2001; Kulak et al., 2002a,b; Han et al., 2003). Furthermore, there are differential changes in nAChRs after MPTP treatment, with a complete loss of 125I-α-conotoxin MII sites and also declines in α-conotoxin MII-resistant 125I-epibatidine sites. Thus, radioligand binding studies suggest that α6β2* and/or α3β2*, as well as α4β2*, nAChRs are present in monkey striatum, with preferential declines in α6β2* and/or α3β2* sites, and smaller losses in α4β2*-expressing receptors with nigrostriatal damage. Treatment with l-DOPA, the most frequently used therapy for Parkinson's disease, also resulted in changes in nAChRs with a selective loss of a low-affinity α-conotoxin MII-sensitive site (Quik et al., 2003a).
The objective of the present study was to further identify the nAChR subunit composition in monkey striatum and the effect of nigrostriatal damage and l-DOPA treatment on the different receptor populations. To approach this, receptor binding studies using nAChR-directed radioligands and immunoprecipitation experiments using subunit-selective antibodies were done in striata from control and treated monkeys.
Materials and Methods
Animals and Treatment
Adult squirrel monkeys (Saimiri sciureus) weighing 0.5 to 0.8 kg were purchased from Osage Research Primates (Osage Beach, MO), and quarantined upon arrival. They were housed in a 13-h/11-h light/dark cycle. They had free access to water and were given food pellets and fruit once daily. All procedures used conform to the National Institutes of Health Guide for the Care and Use of Laboratory Animals and were approved by the Institutional Animal Care and Use Committee. MPTP (2 mg/kg, s.c.) treatment was as described previously (Quik et al., 2000b). To evaluate the behavioral effects of the lesion, animals were rated for parkinsonism using a modified Parkinson rating scale for the squirrel monkey, in which the disability scores ranged from 0 to 20. The composite score was evaluated based on 1) spatial hypokinesia, 2) body bradykinesia, 3) manual dexterity, 4) balance, and 5) freezing. A group of unlesioned animals was administered l-DOPA (15 mg/kg) in combination with carbidopa by oral gavage twice daily, 4 h apart, on a 5-day/2-day on/off schedule for 8 weeks.
Animals were euthanized in accordance with the recommendations of the Panel on Euthanasia of the American Veterinary Medical Association and conforming to the National Institutes of Health Guide for the Care and Use of Laboratory Animals. Ketamine hydrochloride (15–20 mg/kg, i.m.) was administered for sedation, followed by injection of 0.22 ml/kg i.v. euthanasia solution (390 mg/ml of sodium pentobarbital and 50 mg/ml phenytoin sodium). When the heart had stopped, the brains were rapidly removed, placed in a mold, and cut into 6-mm blocks. These were frozen in isopentane on dry ice and stored at -80°C. Striatal and cortical tissue was dissected from half the brain and used for the antibody immunoprecipitation studies. The other half of the brain was used for the autoradiographic studies. Sections (20 μm) were cut using a cryostat, thaw-mounted onto poly-l-lysine–coated slides, air-dried, and stored at -80°C. For the receptor binding studies, MPTP-treated monkeys were separated into two groups as reported previously (Quik et al., 2001; Kulak et al., 2002a). Monkeys with striatal dopamine transporter levels ∼30% of control were defined as moderately lesioned, whereas those with transporter levels ≤5% of control were defined as severely lesioned. Only tissue from severely lesioned animals was used for the immunoprecipitation studies.
Antibody Production and Characterization
The polyclonal antibodies against the human α2, α3, α4, α5, α6, β2, β3, or β4 monkey nAChR peptide subunits (Table 1) were produced in rabbit as described previously (Zoli et al., 2002; Champtiaux et al., 2003) and affinity-purified. The peptides obtained from monkey or human sequences were located in the putative cytoplasmic loop between M3 and M4. The affinity-purified antisera were bound to CNBr-activated Sepharose at a concentration of 1 mg/ml, and the columns used for subtype immunopurification.
Because no cloned nAChR monkey subunits are available, the specificity of the antibodies produced against the human peptides was tested by quantitative immunoprecipitation experiments using extracts obtained from human embryonic kidney cells transfected with different combinations of the human α2, α3, α4, α5, α6, β2, and β4 subunits (a generous gift from Dr. E. Sher of Eli Lilly and Co Ltd, Bristol, UK) or from tissues obtained from wild-type and nAChR-null mutant mice. Triton X-100 (2%) extracts, labeled with 2 nM [3H]epibatidine, prepared from the transfected cells or from tissues obtained from wild-type or knockout animals, were incubated with the saturating concentrations of the antibodies directed against all the subunits. In these tissues, the antibodies recognized only the receptors containing the corresponding subunits. The immunoprecipitation capacity of these antibodies versus the human and rodent subtypes was very high (>80%) (Zoli et al., 2002; Champtiaux et al., 2003; Moretti et al., 2004). The anti-α4, -α6, -β2, and -β3 antibodies were also tested on monkey-purified subtypes (see Results), where they also had a very high immunoprecipitation capacity. Binding values ≤6% were at the detection level of the assay, so this value was used as our cut-off for subunit expression.
Preparation of Membranes and 2% Triton X-100 Extracts from Monkey Brain
Monkey striatum and cortex, obtained as described above, was separately homogenized in an excess of 50 mM sodium phosphate pH 7.4, 1 M NaCl, 2 mM EDTA, 2 mM EGTA, and 2 mM phenylmethylsulfonyl fluoride for 2 min using an UltraTurrax homogenizer. The homogenates were then diluted and centrifuged for 1.5 h at 60,000g. The homogenization, dilution, and centrifugation of the indicated tissue was performed twice, after which the pellets were collected, rapidly rinsed with 50 mM Tris HCl, pH 7, 120 mM NaCl, 5 mM KCl, 1 mM MgCl2, 2.5 mM CaCl2, and 2 mM phenylmethylsulfonyl fluoride and then resuspended in the same buffer containing a mixture of 20 μg/ml of each of the following protease inhibitors: leupeptin, bestatin, pepstatin A, and aprotinin. Triton X-100 at a final concentration of 2% was added to the washed membranes, which were extracted for 2 h at 4°C. The extracts from tissues were then centrifuged for 1.5 h at 60,000g and recovered. An aliquot of the resultant supernatants was collected for protein measurement using the bicinchoninic acid protein assay (Pierce, Rockford, IL) with bovine serum albumin as the standard.
[3H]Epibatidine Binding Assays for Immunoprecipitation Studies
Membrane binding experiments were performed by incubating membrane homogenates overnight with 2 nM [3H]epibatidine (56 Ci/mmol; Amersham Biosciences, Piscataway, NJ) at 4°C. To prevent binding of [3H]epibatidine to α-bungarotoxin-binding receptors, membranes were preincubated with 2 μM α-bungarotoxin and then with [3H]epibatidine. Specific radioligand binding was defined as total binding minus nonspecific binding determined in the presence of 100 nM unlabeled epibatidine. The 2% Triton X-100 extracts of tissues were preincubated with 2 μM α-bungarotoxin for 3 h and then labeled with 2 nM [3H]epibatidine. Tissue extract binding was performed using DE52 ion-exchange resin (Whatman, Maidstone, UK) as described previously (Vailati et al., 1999).
Immunoprecipitation of [3H]Epibatidine-Labeled Receptors by Anti-Subunit–Specific Antibodies
Striatal and cortical extracts or purified receptors were preincubated with 2 μM α-bungarotoxin, labeled with 2 nM [3H]epibatidine, and incubated overnight with a saturating concentration of affinity-purified IgG (20–30 μg; Sigma Chemical, St. Louis). The immunoprecipitation was recovered by incubating the samples with beads containing bound anti-rabbit goat IgG (Technogenetics, Milan, Italy). The level of antibody immunoprecipitation was expressed as the percentage of [3H]epibatidine-labeled receptors immunoprecipitated by the antibodies (taking the amount present in the 2% Triton X-100 extract solution before immunoprecipitation as 100%) or as femtomoles od immunoprecipitated receptors per milligram of protein.
For each purification experiment, the 2% Triton X-100 extract obtained from striatal membranes, prepared as described above, was incubated three times with 5 ml of Sepharose-4B bound anti-α6 antibody to remove the α6* receptors. The flow-through of the α6 column was analyzed for the subunit content of the remaining receptors and then incubated two times with 5 ml of anti-β2 antibody bound to Sepharose-4B. The bound β2* nAChRs were then eluted with the β2 peptide and analyzed for their subunit composition by quantitative immunoprecipitation.
The 2% Triton X-100 cortical extract was incubated with 5 ml of Sepharose-4B bound anti-α4 antibodies to remove the α4 receptors. The bound receptors were eluted by competition with 100 μM concentrations of the corresponding α6 or α4 peptides used for antiserum production.
Receptor Autoradiography
[125I]RTI-121 Autoradiography. [125I]RTI-121 (2200 Ci/mmol; PerkinElmer Life and Analytical Sciences, Boston, MA) was used to measure binding to the dopamine transporter (Quik et al., 2001). Sections were preincubated twice for 15 min each in 50 mM Tris-HCl buffer, pH 7.4, containing 120 mM NaCl and 5 mM KCl. Incubation (2 h) was done in the same buffer plus 0.025% BSA, 1 μM fluoxetine, and 50 pM [125I]RTI-121. The sections were washed four times for 15 min each at 4°C in preincubation buffer, dipped in ice-cold water, air-dried, and placed against Kodak MR film (PerkinElmer Life and Analytical Sciences) for 1 to 3 days with 125I microscale standards (Amersham Biosciences). Nomifensine (100 μM) was used to define nonspecific binding.
125I-Epibatidine Autoradiography.125I-Epibatidine binding to striatal sections was done as described previously (Perry and Kellar, 1995; Kulak et al., 2002a). In brief, sections were preincubated for 30 min, and then incubated for 40 min at room temperature in 50 mM Tris buffer, pH 7, 120 mM NaCl, 5 mM KCl, 2.5 mM CaCl2,and1mM MgCl2, containing 0.015 nM 125I-epibatidine (2200 Ci/mmol; PerkinElmer Life and Analytical Sciences). For competition studies, a concentration range of 10 pM to 10 μM α-conotoxin MII was used. Sections were subsequently washed (4°C) for 5 min with buffer (2×) and for 10 s in ice-cold H2O and then air-dried. They were exposed for 2 to 5 days to Kodak MR film (PerkinElmer Life and Analytical Sciences), together with 125I standards (Amersham Biosciences). Nicotine (10 μM) was used to determine nonspecific binding, which was the same as film blank.
125I-A-85380 Autoradiography. Preparation of 125I-A85380 (specific activity, 1500 Ci/mmol) and binding to brain membranes was perfromed as described previously (Mukhin et al., 2000). Preincubation was for 20 min in the same buffer used for 125I-epibatidine binding assays, followed by a 40-min incubation in fresh buffer containing 125I-A-85380 (80 pM). Sections were washed in buffer at 4°C twice for 5 min each, followed by a 10-s wash in deionized H2O (4°C). Air-dried slides were exposed to Kodak MR film (PerkinElmer Life and Analytical Sciences) for 1 to 2 days with 125I standards (Amersham Biosciences). Nicotine (10 μM) was used to determine nonspecific binding, which was the same as film blank.
[3H]Cytisine Autoradiography. [3H]Cytisine (specific activity, 37.5 Ci/mmol; PerkinElmer Life and Analytical Sciences) binding was performed as described previously (Perry and Kellar, 1995; Sihver et al., 1998). Sections were incubated at room temperature for 60 min in buffer (50 mM Tris, pH 7, 120 mM NaCl, 5 mM KCl, 2.5 mM CaCl2, and 1 mM MgCl2) plus 2 nM [3H]cytisine. After incubation, sections were washed twice for 5 min each in buffer at 4°C and 1 × 10 s in ice-cold H2O. After drying at room temperature, slides were exposed for 8 to 12 weeks to 3H-sensitive Hyperfilm (Amersham), along with 3H standards (American Radiolabeled Chemicals, Inc., St. Louis, MO). Nicotine (10 μM) was used to determine nonspecific binding.
125I-α-Conotoxin MII Autoradiography.125I-α-conotoxin MII (specific activity, 2200 Ci/mmol) was synthesized and radiolabeled as described previously (Whiteaker et al., 2000). For assay (Whiteaker et al., 2000; Quik et al., 2001), sections were preincubated at room temperature for 15 min in binding buffer (144 mM NaCl, 1.5 mM KCl, 2 mM CaCl2, 1 mM MgSO4, 20 mM HEPES, and 0.1% BSA, pH 7.5) plus 1 mM phenylmethylsulfonyl fluoride. This was followed by a 1-h incubation at room temperature in binding buffer plus 0.5% BSA, also containing 5 mM EDTA, 5 mM EGTA, and 10 μg/ml each of aprotinin, leupeptin, and pepstatin A, and 0.5 nM 125I-α-conotoxin MII. To terminate the assay, slides were rinsed for 30 s in binding buffer at room temperature followed by 30 s in ice-cold Buffer, two 5-s rinses in 0.1× binding buffer (0°C) and two washes in water (0°C). The sections were air-dried and exposed to Kodak MR film (PerkinElmer Life and Analytical Sciences) for 2 to 5 days together with 125I-standards (Amersham Biosciences). Epibatidine (0.1 μM) was used to determine nonspecific binding.
125I-α-Bungarotoxin Autoradiography. Sections were preincubated at room temperature in 50 mM Tris HCl, pH 7, for 30 min (Clarke and Pert, 1985). They were next incubated for 1 h in the same buffer containing 3 nM 125I-α-bungarotoxin (specific activity 128 Ci/mmol, PerkinElmer Life and Analytical Sciences). The sections were then rinsed four times for 15 min each in ice-cold buffer and once in ice-cold water, air-dried, and placed against Kodak MR film for 1 to 2 weeks (PerkinElmer Life and Analytical Sciences). Nicotine (100 μM) was used to define nonspecific binding.
Analyses of Autoradiographic Data. A squirrel monkey (Saimiri sciureus) brain atlas was used to identify brain regions, as described previously (Quik et al., 2000a). The optical density values, determined using an ImageQuant system (Amersham Biosciences), were assessed by subtracting background from tissue values. This was followed by conversion to femtomoles per milligram of tissue using standard curves generated from radioactive standards simultaneously exposed to the films. Sample optical density readings were within the linear range of the film. Receptor binding data for any one animal represents the mean from one to two sections each from two or more independent experiments.
Competition curves were compared and best-fit to one- and two-site models using Prism (GraphPad Software, San Diego, CA). Statistical analyses were done using one-way analysis of variance followed by Newman-Keuls multiple comparison test where p ≤ 0.05 was considered significant. All values are expressed as the mean ± S.E.M. of the indicated number of animals.
Results
Characterization of nAChR Subunit Antibodies. The identification of nAChR subtypes in monkey brain relied on the use of a series of antisera raised against unique amino acid sequences of the different human or monkey subunits. All of the antibodies (except for the anti-β3 antibody, which was not tested) selectively interacted with receptors expressing the appropriate human nAChR subunit in transfected HEK cells. Because of the sequence identity between α3 and α6 subunits, we also tested whether identification of α3* nAChRs (14%) in striatum might be caused by cross-reactivity of the anti-α3 antibodies with α6* receptors; however, the α3 antibody recognized only 3% of purified α6* receptors. In addition, the immunoprecipitation capacity and specificity of the antibodies was investigated on purified α6* receptors obtained from striatum and on α4* receptors purified from the cortex. We found that the α4, α6, β2, and β3 antibodies had an immunoprecipitation capacity of more than 60%. We did not consider the contribution of subunits to receptor composition that were immunodetected in amounts of 6% or less, and therefore minor nAChR subtypes may have been excluded from the analyses.
NAChR Subunit Expression in Control Monkey Striatum and Cortex. Experiments were first done to quantify the relative contribution of each nicotinic subunit to [3H]epibatidine binding present in the striatum. To approach this, we performed quantitative immunoprecipitation experiments using subunit-specific antibodies and [3H]epibatidine-labeled receptors. Receptor levels in control monkey striatum were 55.5 ± 4.1 and 69.6 ± 5.5 fmol/mg of protein in the membrane preparation and 2% Triton extract, respectively. The receptors immunoprecipitated by specific nAChR subunit antibodies (calculated as the percentage of the total number of [3H]epibatidine receptors) were: β2 (91%), α4 (55%), α6 (25%), β3 (18%), α3 (14%), and α2 (12%) (Fig. 1A). The α5 and β4 subunit containing receptors fell below the detection limit of the assay (6%). Values represent the mean ± S.E.M. of six immunoprecipitation experiments performed in duplicate for each antibody.
A similar approach using [3H]epibatidine-labeled sites was used to identify the major nAChR subtypes in monkey cortex. Receptor levels in control monkey cortex were 41.6 ± 3.6 and 49.9 ± 2.6 fmol/mg of protein in the membrane preparation and 2% Triton extract, respectively. Immunoprecipitation studies using crude membrane extracts showed that receptors contained the β2 (96%), α4 (77%), α2 (21%), and α3 (10%) subunits, whereas the α5, α6, β3, and β4 subunits were below the level of detection of the assay. Results represent the mean ± S.E.M. of three immunoprecipitation experiments performed in duplicate for each antibody (Fig. 1B).
Thus, similar to the rodent, the major nicotinic receptor subtypes in monkey cortex contain the α4 and β2 subunits, whereas in the striatum, they contain α4, α6, β2, and β3 subunits. On the other hand, the α2 and α3, but not α5 and β4, subunits are present in monkey striatum and cortex, distinct from rodent brain.
Subunit Composition of α6* nAChRs in Monkey Striatum. Our immunoprecipitation experiments, as well as previous receptor studies, indicate that there is a selective expression of α6* nAChRs in monkey striatum. To identify the subunits that coassemble with α6, we immunodepleted striatal extract of α6* receptors using an affinity column with a bound anti-α6 antibody. Selective α6* nAChR immunodepletion was confirmed by the fact that immunoprecipitated α6* [3H]epibatidine-labeled receptors decreased from 25% in the total striatal extract to 1% in the flow-through of the α6 column. In addition, α4* receptors were increased (from 58.5 to 71.6%), suggesting that an appreciable portion of the α4 subunit pool is not assembled with the α6 subunit. α2* nAChRs were also substantially increased in the flow through (from 12 to 30%), suggesting they may primarily be associated with non-α6* nAChRs. On the other hand, β3* receptors markedly decreased suggesting a colocalization with α6. β2* nAChRs remained unchanged indicating they are present in the majority of receptor subtypes.
To identify their subunit composition, α6* receptors were eluted from the affinity column with α6 peptide and labeled with [3H]epibatidine; the eluate was immunoprecipitated with nAChR subunit-specific antisera. As shown in Fig. 1C, the anti-α4, -β2, and -β3 sera immunoprecipitated 47, 100, and 61% of the purified [3H]epibatidine-labeled receptors, respectively. In contrast, the anti-α2, -α3, -α5, and -β4 sera immunoprecipitated ≤6% (detection limit of the assay) of [3H]epibatidine binding, suggesting they do not coassemble with α6.
The dual immunoprecipitation data suggest that α6* nAChRs may be composed of α6β2β3 and/or α6α4β2β3 subunits. In addition, analyses of the α6-affinity column flow-through indicate that α4β2* nAChRs also form major striatal subtypes.
Subunit Composition of non-α6* nAChRs in Monkey Striatum. To identify striatal nAChRs not containing the α6 subunit, we also immunopurified the flow-through of the α6 affinity column using an anti-β2 column. We then eluted the bound receptors with β2 peptide and performed immunoprecipitation studies using subunit specific antisera. The anti α2, α3, and α4 antibodies immunoprecipitated 22.9 ± 3.9, 20.4 ± 5.6, and 73.4 ± 2.4% (mean ± S.E.M., n = 2) of the [3H]epibatidine-labeled purified β2* receptors, respectively. The other antibodies yielded no detectable immunoreactive material.
These studies clearly show that, in addition to α6* nAChRs, α4β2* receptors are also present in monkey striatum together with a minor population of α2β2* and α3β2* nAChRs. Because of the low recovery of the α2* and α3* subtypes in the β2 purified receptor preparation, it was not feasible to further investigate their subunit composition.
Subunit Composition of α4* nAChRs in Monkey Cortex. Because α4 is the major acetylcholine binding subunit in cortex, experiments were done to determine with which subunits α4 is coexpressed (Fig. 1D). Cortical extracts were incubated with anti-α4 antibody linked to Sepharose beads. Bound α4* receptors were then eluted with α4 peptide. Immunoprecipitation experiments showed that 95% of these receptors contained the β2 subunit, 17% the α2 subunit, and 8% the α3 subunit. Therefore, all α4* receptors most likely couple with β2, whereas a subpopulation of α4β2* subtypes also contain the α2 and α3 subunits.
Nigrostriatal Damage Decreases Select nAChR Subunits in Monkey Striatum. Studies were next done to determine the effect of nigrostriatal damage on nAChR subunit expression in monkey striatum (Table 2). Animals were lesioned with the selective dopaminergic neurotoxin MPTP and euthanized 1 month later when the effects of the lesion were maximal. [3H]epibatidine binding in monkey striatum was significantly (p < 0.005) reduced from 55.5 ± 4.1 to 30.0 ± 3.5 fmol/mg of protein (n = 6 experiments) in the membrane preparation and from 69.6 ± 5.5 to 35.2 ± 1 fmol/mg of protein in the 2% Triton extract (n = 6 experiments), similar to previous results (Kulak et al., 2002a). Immunoprecipitation of solubilized [3H]epibatidine binding sites using subunit-specific antibodies (Table 2) showed that MPTP-lesioning produced the greatest decline (expressed as percentage decrease) in α6* (83%) and β3* (86%) subtypes, as well as significant reductions in receptors containing α3 (50%), α4 (32%), and β2 (48%) subunits but not α2 subunits. Because α6* and β3* nAChRs were decreased in parallel with the dopamine transporter, these subtypes are most likely coexpressed on dopamine terminals. In contrast, receptors expressing the α4 and β2 subunits seem to be present on both dopaminergic and nondopaminergic neurons, whereas α2* subtypes are on nondopaminergic cells. Putative receptor subtypes in striatum thus include α6β2β3, α6a4β2b3, α4β2, and α2β2*.
Consistent with previous results, cortical [3H]epibatidine receptors were unaffected by MPTP-treatment with 41.6 ± 3.6 and 41.4 ± 5.8 fmol/mg of protein in membranes from control animals and MPTP-treated animals, respectively. The 2% Triton extracts were also similar in controls and MPTP treated animals with values of 49.9 ± 2.7 and 47.3 ± 0.8 fmol/mg of protein, respectively. Immunoprecipitation studies performed on cortical tissues confirmed that there was no change in the expressed subtypes after MPTP lesioning.
Radioligand Binding Studies—Effect of Nigrostriatal Damage. Earlier work had shown that receptors labeled with 125I-epibatidine, a ligand that identifies multiple receptor subtypes (α2* through α6*) were reduced with nigrostriatal damage (Kulak et al., 2002a), consistent with the present immunoprecipitation data. Other studies using the more selective radioligand 125I-α-conotoxin MII further demonstrated specific declines with lesioning in α3* and/or α6* nAChRs (Quik et al., 2001). In the present experiments, we investigated binding of 125I-α-bungarotoxin to α7 receptors and [3H]cytisine, which interacts with α4β2* and α2β2* subtypes (Luetje and Patrick, 1991). Autoradiographic studies showed there was a decrease in [3H]cytisine binding in caudate and putamen (Fig. 2A) but no change in 125I-α-bungarotoxin binding (Fig. 2B).
Previous work in rodents had indicated that [3H]cytisine binds to an α4* nAChR (Flores et al., 1992). The present results (Fig. 3A) show that α-conotoxin MII does not compete with [3H]cytisine in striatal slices from either control or MPTP-lesioned animals. This observation suggests that [3H]cytisine binds at a similar receptor interface (that is, α4β2) in monkey striatum. Nicotine completely blocked [3H]cytisine binding in striatum from both control and MPTP-lesioned monkeys (Fig. 3B), demonstrating that the radioligand binds to a receptor with nicotinic characteristics. Previous studies (Quik et al., 2001) had shown that the nAChRs decreased with nigrostriatal damage were α-conotoxin MII-sensitive (that is, α3* and/or α6*). This work, combined with the present experiments showing that [3H]cytisine binding (α4*) receptors are decreased after MPTP treatment (Fig. 4), suggests that these nAChRs may have both an α4β2 and an α6β2 interface (that is, α6β2α4β2*).
l-DOPA Treatment Decreases nAChRs in Monkey Striatum. Previous studies had shown that 2 weeks of l-DOPA treatment (15 mg/kg twice daily, every 4 h) reduced striatal 125I-epibatidine sites (Quik et al., 2003a). To determine whether a longer course of treatment might result in a differential decline, we investigated the effect of 8 weeks of administration. Results (Fig. 5A) show that there was a somewhat greater decline in 125I-epibatidine binding (∼25%), with similar results obtained using 125I-A85380. No change was observed in 125I-α-conotoxin MII binding sites or [125I]RTI-121 binding to the dopamine transporter.
Competition studies of 125I-epibatidine binding by α-conotoxin MII were then done to determine whether l-DOPA treatment had selective effects on different nAChR populations after 8 weeks of treatment. Analyses of the inhibition curves demonstrated a biphasic α-conotoxin MII inhibition of striatal 125I-epibatidine binding in control animals but not in l-DOPA–treated animals. The control data best fit to a two-site competition model with IC50 values of 1.78 nM (CI 0.7 to 4.0 nM) and 1.14 μM (CI 0.10 to 9.0 μM), whereas the data from the treated animals fit best to a one-site competition model with an IC50 value of 8.37 nM (CI 2.4 to 28 nM). Thus, 8 weeks of l-DOPA treatment led to a selective decrease in low-affinity but not high-affinity α-conotoxin MII–sensitive sites consistent with the lack of change in 125I-α-conotoxin MII (Fig. 5B).
As an approach to understand the subunit composition of the striatal nAChR sites affected by l-DOPA treatment, immunoprecipitation studies were done (Fig. 5C). No significant declines were observed in nAChR subunit-immunoreactivity with l-DOPA treatment compared with control animals.
Discussion
Using a combined molecular and pharmacological approach, we investigated nAChR subunit composition in striatum using control and MPTP-lesioned monkeys. The results show that several major populations are present in striatum including α7, α4β2*, α6β2*, α3β2*, and α2β2* nAChRs. Detailed analyses of the present data, combined with previous receptor binding and recent functional studies suggest the following: 1) α6β2* nAChRs contain β3 and also, in part, α4 to form α6β2β3 and α4α6β2β3 subtypes, 2) the presence of striatal α4β2 and α2β2* nAChRs, and 3) the existence of a novel α3β2* nAChR population. A detailed rationale for the existence of these subtypes and their localization (Fig. 6) in monkey striatum is discussed below.
Receptor Subtypes Present in Monkey Striatum. Our postulated composition of striatal nAChR subtypes is based on the current hypothesis that heteromeric nAChRs have at least two subunits bearing the principal amino acid loops for acetylcholine binding interfaces (α2, α3, α4, or α6 subunits) and two subunits bearing the complementary amino acid loops (β2 or β4 subunits), whereas the fifth subunit can be either a complementary or a purely structural subunit (α5 or β3 subunits).
Receptor Subtypes Present on Striatal Dopaminergic Terminals—α6α4β2β3, α6β2β3, α3β2*. Our previous data had shown that nigrostriatal damage leads to a selective decline in striatal nAChRs that bind 125I-α-conotoxin MII, a ligand that interacts at an α3β2* and/or α6β2* interface, with no change in other receptor subtypes (McIntosh et al., 1999; Quik et al., 2001; Kulak et al., 2002a; Nicke et al., 2004). These findings suggested that receptors expressing α6β2 and/or α3β2 subunits are localized to dopaminergic terminals in monkey striatum. The present results show that [3H]cytisine, a ligand that interacts at an α4β2 receptor interface (Flores et al., 1992), binds to monkey striatum and, in addition, that [3H]cytisine binding is reduced with moderate nigrostriatal damage. Previous data using 125I-epibatidine had shown that a moderate lesion decreased only α-conotoxin MII-sensitive nAChRs (Quik et al., 2001; Kulak et al., 2002a). These combined data can most readily be explained by postulating the existence of a receptor subtype with both an α6β2 and also an α4β2 interface (that is, an α6β2α4β2* subtype).
The current antibody experiments support and extend the results from the receptor studies. The dual immunoprecipitation shows that all striatal α6-subunit-immunoreactivity is precipitated by the anti-β2 antibody, suggesting an absolute requirement for an α6β2 interface, in agreement with the 125I-α-conotoxin MII binding data. In addition, the lesion studies show that the α6 and β3 subunit are decreased in parallel after nigrostriatal damage, suggesting they are coexpressed, thus forming an α6β2β3* receptor. The anti-α4 but not the anti-α2 and anti-α3 antibodies also immunoprecipitated α6* nAChRs, whereas the α5 and β4 subunits were not detectable in striatum. Together, these observations reduce the potential subunit combinations to α6β2β3 and α6α4β2β3. These subtypes may both be present in striatum, because the anti-α4 antibody only precipitated a portion of the α6* sites.
The immunoprecipitation data also show that α3 subunit-immunoreactivity is present in striatal extracts. Furthermore, our studies using a purified β2* receptor preparation clearly show that the α3 and β2 subunit coprecipitate. These results provide direct evidence that α-conotoxin MII binds at an α3β2 interface in monkey striatum, as suggested previously (McIntosh et al., 1999; Kulak et al., 2002b; Nicke et al., 2004). Lesion studies show that all α-conotoxin MII-sensitive receptors are lost with nigrostriatal damage, suggesting that they are present on striatal dopaminergic terminals. These combined data suggest that α3β2* nicotinic receptors are located on nigrostriatal terminals in monkey brain, together with the α6α4β2β3 and α6β2β3 subtypes.
Receptor Subtypes Present on Dopaminergic and Nondopaminergic Striatal Neurons—α4β2 and α2β2*. As discussed earlier, results show that 30% of the [3H]cytisine sites (containing an α4β2 interface) are decreased with moderate nigrostriatal damage, suggesting they form an α6β2α4β2β3 subtype. The remaining [3H]cytisine binding sites would represent non-α6 α4β2* nAChRs, which may be both pre- and postsynaptic. The presence of this latter population is also confirmed from the results of the dual label β2 immunoprecipitation experiments using the non-α6* receptor preparation. Evidence for a presynaptic localization for a portion of the α4β2* receptors stems from the results of our functional studies showing that ∼30% of nicotine-evoked [3H]dopamine release from striatal synaptosomes is resistant to inhibition by α-conotoxin MII (McCallum et al., 2004).
The immunoprecipitation data are consistent with these findings and allow us to speculate as to the remaining composition of the α4β2* sites. They do not seem to contain α3 or α6 because they are α-conotoxin MII-resistant. They are also most probably not expressed with the β3 subunit because the lesion studies indicate that β3 is coexpressed with α6. The absence of the α5 and β4 subunits in monkey striatum rules out their presence in the α4β2* pentamer. Thus, the only remaining subunit that can form a receptor with α4β2* receptors is α2, yielding α4β2 and α4α2β2 nAChRs. This finding is supported by our studies using total striatal extracts and a non-α6 containing β2 purified receptor preparation, which showed that a large proportion of β2* receptors contain the α4 subunit, and a minority contained the α2 subunit.
The α4 and α2 subunits may be present within the same or on distinct nAChR subtypes, allowing for the presence of α4β2 and α2β2* nAChRs. Because α2 is not affected by nigrostriatal damage, the α2β2* receptors are most likely to be found on nondopaminergic neurons, as in the rodent (Zoli et al., 2002). In summary, dopaminergic terminals exclusively express α4β2 receptors, whereas α4β2 and α2β2* receptors may be expressed on nondopaminergic neurons.
Receptors Present Exclusively on Nondopaminergic Striatal Elements. The 125I-α-bungarotoxin binding studies show that striatal α7 receptor expression is relatively low and unaffected by nigrostriatal damage. These data suggest that these sites are localized on striatal GABA-ergic and cholinergic neurons, glutamatergic inputs, and/or nonneuronal cells (Kaiser and Wonnacott, 2000; Rogers et al., 2001). With respect to number of binding sites per receptor, homomeric α7 nAChRs are likely to have five acetylcholine sites, whereas heteromeric receptors with several different α subunits contain at least two binding sites and possibly more depending on the nature of the other α subunits.
l-DOPA Treatment Differentially Affects Striatal Nicotinic Receptors. Previous studies had shown that a 2-week treatment with l-DOPA, a commonly used therapy for Parkinson's disease, resulted in a ∼20% decline in striatal α-conotoxin MII–sensitive 125I-epibatidine sites with no change in 125I-α-conotoxin MII binding (Quik et al., 2003a). Because patients are treated with l-DOPA for extended times, we next investigated the effect of an 8-week treatment course. The results show that the decline in striatal α-conotoxin MII–sensitive 125I-epibatidine sites persists with continued l-DOPA treatment. The finding that there is no change in binding of 125I-α-conotoxin MII (0.5 nM) to high-affinity sites, suggests a preferential loss in low- but not high-affinity α-conotoxin MII–sensitive receptors. This is supported by the competition data, which best fit to a one-site model after l-DOPA treatment but to a two-site model in the control condition.
These data are in apparent contradiction with the immunoprecipitation results, which show no significant difference in nAChR subunit-immunoreactivity in animals treated with l-DOPA compared with control. These results may suggest that l-DOPA treatment induces a change/redistribution in composition of α3* and/or α6* nAChR subtypes (as detected in the radioligand binding assays) without affecting the total amount of these two subunit (as measured by the immunoprecipitation assay). On the other hand, or as well, the varying results between the two assays may reflect a greater sensitivity of the autoradiographic binding technique compared with immunoprecipitation.
Receptor Subtypes Present in Monkey Cortex. The major nAChR receptor populations in cortex seem to contain α4β2 subunits, in agreement with previous studies in rodents (Flores et al., 1992; Zoli et al., 2002; Champtiaux et al., 2003). In contrast, α2* nAChRs were also identified in monkey cortex, an observation consistent with the identification of α2 mRNA in monkey brain (Han et al., 2003). Both α4β2 and α4β2α2 nAChRs seem to be present with this latter subtype representing ∼16% of the α4β2* cortical receptor population. We also identified α3* receptors in monkey cortex (8%), an observation consistent with recent findings demonstrating the presence of α3β2* and/or α6β2* nAChRs in human cortex (Amtage et al., 2004; Quik et al., 2004). Neither MPTP-lesioning nor l-DOPA treatments affected cortical nAChRs, as previously shown (Kulak et al., 2002a; Quik et al., 2003a).
Summary. The present results show that several major nAChR populations are present in monkey brain. In cortex, we identified α7 and α4β2 subtypes and also novel nAChR populations expressing α4β2α2 and α3β2* subunits. In striatum, α7, α4β2, α6β2* (α6β2β3 and α4α6β2β3), and α2β2* subtypes were identified in agreement with rodent studies, as well as the α3β2* subtype that is distinct from rodent brain.
Acknowledgments
We thank Dr. Emanuele Sher (Eli Lilly and Co Ltd, UK) for the generous gift of membranes of transfected α2β4, α3β4, α4β4 α3β2, α3β2, α3α5β2, α4α6β4, and α3α6β4 cells.
Footnotes
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This work was supported in part by the California Tobacco-Related Disease Research Program grant 11RT-0216, by National Institutes of Health (NIH) grants NS42091 and NS47162 (to M.Q.), by Italian Ministero dell'Istruzione, dell'Università e della Ricerca grant MM05152538, Italian Ministry of Health grant ICS 030.3/RA 0048, European Research Training Network HPRN-CT-2002-00258, the Fondo Integrativo Speciale per la Ricerca-Consiglio Nazionale delle Ricerche Neurobiotecnologia 2003, the Fondazione Cariplo grant 2002/2010 (to F.C.), Fondo per gli Investimenti della Ricerca di Base grant RBNE01RHZM 2003 (to C.G.), and NIH grants MH53631 and DA12242 (to J.M.M.).
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S.V. and T.B. contributed equally to the work.
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Article, publication date, and citation information can be found at http://molpharm.aspetjournals.org.
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doi: 10.1124/mol.104.006015.
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ABBREVIATIONS: nAChR, nicotinic acetylcholine receptor; MPTP, 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine; A85380, 3-(2(S)-azetidinylmethoxy)pyridine dihydrochloride; RTI-121, 3β-(4-iodophenyl)tropane-2β-carboxylic acid isopropyl ester; BSA, bovine serum albumin; CI, confidence interval; *, nicotinic receptors containing the indicated α and/or β subunit and also additional undefined subunits.
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↵1 Present address: Department of Pathology, Anatomy, and Cell Biology, Thomas Jefferson University, Philadelphia, Pennsylvania.
- Received August 9, 2004.
- Accepted October 5, 2004.
- The American Society for Pharmacology and Experimental Therapeutics