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Institut National de la Santé et de la Recherche Médicale Unité 679, Experimental Neurology and Therapeutics, Hpital de la Salpêtrière, Paris, France
Abstract
We evaluated the neuroprotective potential of tachykinin peptides using a model system in which mesencephalic dopaminergic (DA) neurons die spontaneously and selectively as they mature. The three native tachykinins, substance P (SP), neurokinin (NK) A, and NKB afforded substantial protection against DA cell demise. The selective NK1 receptor antagonist [D-Pro9,[spiro--lactam] Leu10,Trp11]substance P (GR71251) was sufficient in itself to suppress the effect of SP, whereas a cotreatment with GR71251 and the NK3 receptor antagonist (R)-N-[-(methoxycarbonyl)benzyl]-2-phenylquinoline-4-carboxamide (SB218795) was required to prevent the effects of both NKA and NKB. Consistent with these results, D-Ala-[L-Pro9,Me-Leu8]substance P(7–11) (GR73632), a selective agonist of NK1 receptors and [pro7]-NKB, a selective agonist of NK3 receptors, conferred protection to DA neurons, whereas (Lys3, Gly8-R--lactam-Leu9)neurokinin A(3–10) (GR64349), which activates specifically NK2 receptors, did not. DA neurons rescued by tachykinins accumulated [3H]DA efficiently, which suggests that they were also totally functional. Neuroprotection by tachykinins was highly selective for DA neurons, rapidly reversed upon treatment withdrawal, and reproduced by but independent of glial cell line-derived neurotrophic factor. Survival promotion by tachykinins was abolished by blocking voltage-gated Na+ channels with tetrodotoxin or N-type voltage-gated Ca2+ channels with -conotoxin-MVIIA, which indicates that an increase in neuronal excitability was crucially involved in this effect. Together, these data further support the notion that the survival of mesencephalic DA neurons during development depends largely on excitatory inputs, which may be provided in part by tachykinins.
Tachykinins belong to a family of neuropeptides that share a common C-terminal sequence, Phe-Xaa-Gly-Leu-Met-NH2, that is crucial for interaction with their receptors (Severini et al., 2002; Pennefather et al., 2004). The mammalian tachykinins include substance P (SP), neurokinin (NK) A, and NKB, the effects of which are mediated by the receptors NK1, NK2, and NK3. SP is the most potent tachykinin at the NK1 receptor site, whereas NKA exhibits the highest affinity for the NK2 receptor and NKB for the NK3 receptor (Pennefather et al., 2004). The effects through NK receptors are generally coupled to a G protein (Gq/G11)-regulated phosphoinositide pathway (Khawaja and Rogers, 1996), but G protein-independent coupling has also been described previously (Yang et al., 2003).
NK receptors are implicated in various biological effects that include smooth muscle contraction, inflammatory processes, hypotensive effects, and stimulation of gland secretion (Severini et al., 2002). In the nervous system, tachykinins also operate as neuromodulators/neurotransmitters via mechanisms that are generally excitatory (Stacey et al., 2002). In addition, there is evidence that tachykinins also have intrinsic neuroprotective properties. They were found to reverse -amyloid-induced toxicity (Kowall et al., 1991), to protect against excitotoxic cell death (Calvo et al., 1996), and to limit neurodegeneration caused by trophic factor deprivation (Lallemend et al., 2003).
The loss of nigrostriatal dopaminergic (DA) neurons in Parkinson's disease and related disorders leads to profound motor impairment (Agid, 1991). The identification of signals or factors that control the survival and function of DA neurons is therefore of interest because it might not only provide a better understanding of the pathophysiological mechanisms of these diseases but also foster the development of new therapeutic strategies to halt their progression (Dawson and Dawson, 2002). There is indirect evidence that tachykinins may influence the survival of mesencephalic DA neurons: 1) in the brain, the substantia nigra is the richest area in SP and SP-containing axon terminals (Hokfelt et al., 1991); 2) striatal DA depletion in Parkinson's disease and in animal models of the disease results in profound changes in SP expression (Mauborgne et al., 1983; Betarbet and Greenamyre, 2004); and 3) excitatory stimuli, which are believed to play a key role in the survival of DA neurons during development and possibly in the adult brain (Douhou et al., 2001; Katsuki et al., 2001; Salthun-Lassalle et al., 2004), can be evoked by direct application of NK1 or NK3 agonists to mesencephalic DA neurons (Nalivaiko et al., 1997).
To examine the potential of tachykinins to promote the survival of DA neurons, we have used a model system in which these neurons die spontaneously and progressively as a function of time (Michel and Agid, 1996). More specifically, we wished to evaluate the neuroprotective potential of native and synthetic tachykinins for DA neurons; determine what receptor subtypes are involved; establish whether these mechanisms are shared with or related to that of GDNF, a prototypical neuroprotective factor for DA neurons (Choi-Lundberg et al., 1997); and explore the mechanisms underlying neuroprotection.
Materials and Methods
Peptide Agonists/Antagonists of Tachykinin Receptors and Other Pharmacological Agents. Native tachykinins SP, NKA, NKB, and synthetic peptide agonists/antagonists (GR73632, GR64349, [pro7]-NKB, GR71251, and R396) of tachykinin receptors were from NeoMPS (Strasbourg, France). The nonpeptide receptor antagonist SB218795 was from Bioblock Scientific (Strasbourg, France). Glial cell line-derived neurotrophic factor (GDNF) was purchased from AbCys (Paris, France), and the anti-GDNF neutralizing antibody (AB 212 NA) was from R&D Systems Europe (Lille, France). The pancaspase inhibitor N-tert-butoxycarbonyl-D-fluoromethylketone (Boc-D-FMK) was obtained from Calbiochem (Darmstadt, Germany). Others pharmacological reagents were from Sigma/RBI (Natick, MA). Tritiated neurotransmitters and tritiated methyl-thymidine were from GE Healthcare (Little Chalfont, Buckinghamshire, UK).
Mesencephalic Cell Cultures. Animals were treated in accordance with the Guide for the Care and Use of Laboratory Animals (National Research Council, 1996), the European Directive number 86/609, and the guidelines of the local institutional animal care and use committee. Cultures were prepared from the ventral mesencephalon of embryonic day 15.5 Wistar rat embryos (Janvier Breeding Center, Le Genest St Isle, France) as described previously (Michel and Agid, 1996). After dissection, pieces of mesencephalic tissue were dissociated mechanically with no enzymatic treatment using a Pipetman (Gilson, Villier Le Bel, France) set to 900 μl and plated onto polyethylenimine (1 mg/ml; Sigma-Aldrich, St. Quentin Fallavier, France)-coated 24-well culture plates. Cells plated at a density of 1.5 to 2.0 x 105/cm2 were then maintained at 37°C in a humidified incubator with a 5% CO2 atmosphere using 500 μl of N5 culture medium (Salthun-Lassalle et al., 2004) supplemented with 5 mM glucose, 5% horse serum, and 0.5% fetal bovine serum, except for the first 3 days, when 2.5% fetal bovine serum was used to enhance cell attachment. The cultures were fed daily by replacing 350 μl of the culture medium. They were then supplemented with appropriate pharmacological treatments, including native tachykinins and agonists/antagonists of tachykinin receptors.
Quantification of Neuronal Survival. The survival of DA neurons was quantified by counting the number of cells labeled with an antibody against tyrosine hydroxylase (TH), as described previously. In brief, the cultures were fixed with 4% formaldehyde in phosphate-buffered saline (PBS) for 15 min. Cells were washed three times with PBS and then incubated for 24 h at 4°C with a monoclonal anti-TH antibody (Diasorin, Stillwater, MN) diluted 1:5000 in PBS containing 0.2% Triton X-100. The TH antibody was then revealed with an anti-mouse IgG Cy3 conjugate (1:500; Sigma/RBI) for 2 h at room temperature. Mesencephalic cultures contained between 1 and 2% TH+ cells at the time of plating. All neurons, regardless of their neurotransmitter phenotype, were identified by labeling microtubule-associated protein-2 (MAP2) with a monoclonal antibody (AP-20, Sigma/RBI) diluted 1:100 in PBS revealed with a anti-mouse Alexa Fluor 488 conjugate.
Measurement of Neurotransmitter Uptake. The functional integrity of DA neurons was evaluated by their ability to take up DA by active transport (Douhou et al., 2001). Uptake was initiated by the addition of 50 nM [3H]DA (40 Ci/mmol) to cultures preincubated for 10 min in 500 μl of PBS containing 5 mM glucose and 100 μM ascorbic acid. It was terminated after 15 min by two rapid washes with cold PBS. Cells were scraped off the culture wells, and the accumulated tritium was counted by liquid scintillation spectrometry. The uptake of [3H]DA was also visualized by microautoradiography (Troadec et al., 2002). In this case, the incubation time was prolonged to 30 min, and [3H]DA was used at 100 nM to improve detection. The cultures were then fixed with a mixture of glutaraldehyde/formaldehyde (0.5:4%) and dehydrated with ethanol. Incorporation of [3H]DA was detected with the Hypercoat LM-1 emulsion (GE Healthcare) after an exposure of 10 days in the dark at 4°C. In both paradigms, blank values were obtained in the presence of 0.5 μM GBR-12909 (Sigma-Aldrich, St. Louis, MO). GABA and serotonin (5-HT) uptake was measured as described using 50 nM [3H]GABA and 20 nM [3H]5-HT, respectively (Salthun-Lassalle et al., 2004). Note that the concentrations of the three neurotransmitters were lower than the Km values of their transporters to be within the linear portion of the uptake time course.
Quantification and Identification of Proliferating Cells. [methyl-3H]Thymidine, a marker of DNA synthesis, was used to label and quantify proliferating cells as described previously (Mourlevat et al., 2003). Mesencephalic cultures maintained up to 7 DIV in the presence of test treatments were exposed to 1 μCi of [methyl-3H]thymidine (GE Healthcare; 40 Ci/mmol) for 3 h at 37°C in serum-free N5 medium supplemented with 5 mM glucose. After three rapid washes, the cells were allowed to recover for 1 h in the same culture medium to remove unincorporated radioactivity. The cultures were fixed in 4% formaldehyde for 15 min and when necessary were processed for TH immunofluorescence detection subsequently. They were then dehydrated in ethanol and exposed to Hypercoat LM-1 emulsion (GE Healthcare) for 4 days at 4°C to detect the tritiated label.
Quantification of Intracellular Calcium Levels. Cytoplasmic-free calcium levels were measured in individual neurons using Calcium Green-1-AM (Molecular Probes, Eugene, OR) as described previously (Douhou et al., 2001; Salthun-Lassalle et al., 2004).
Reverse Transcription and Polymerase Chain Reaction. Total RNA from mesencephalic cultures or from rat uterus tissue was isolated with RNABle solution (Laboratories Eurobio, Les Ulis, France). Three types of cultures were used: mixed cultures containing neurons and glial cells, pure neuronal cultures in which glial cells were eliminated by treatment with 3 μM araC, and pure astrocyte cultures. First-strand cDNA was synthesized from 2 μg of total RNA using the RT kit (QIAGEN S.A., Courtaboeuf, France). To analyze the expression of NK1, NK2, and NK3 receptors, transcripts were amplified by polymerase chain reaction under the following conditions: 35 cycles of 94°C for 30 s, 58°C for 30 s, and 72°C for 30 s, with 3 μl of the reverse transcription mixture. The primers used for tachykinin receptors were: NK1: forward, (5'-CCTCCTGCCCTACATCAACCC-3'); and reverse, (5'-CTGTGTCTGGAGGTATCGGG-3'); NK2: forward, (5'-CATCACTGTGGACGAGGGGG-3'); and reverse, (5'-TGTCTTCCTCAGTTGGTGTC-3'); and NK3: forward, (5'-CATTCTCACTGCGATCTACC-3'); and reverse (5'-CTTCTTGCGGCTGGATTTGG-3') (Pinto et al., 1999). The polymerase chain reaction products were visualized by electrophoresis in a 2% agarose gel containing 0.1 mg/ml ethidium bromide.
Statistical Analysis. Simple comparisons between two groups were performed with Student's t test. Multiple comparisons against a single reference group were made by one-way analysis of variance followed by Dunnett's test. When all pairwise comparisons were made, the Student-Newman-Keuls test was used. S.E.M. values were derived from at least three independent experiments.
Results
Native Tachykinins SP, NKA, and NKB Increase the Number of DA Neurons in Mesencephalic Cultures. The native tachykinins SP, NKA, and NKB increased the number of TH+ DA neurons in mesencephalic cultures (Fig. 1, A–C and E). The effects of SP and NKB on the number of TH+ cells were restricted to a narrow range of concentrations between 1 and 50 nM and 1 and 5 nM, respectively, with peak effects at 10 and 1 nM (Fig. 1, A and C). At higher concentrations, no effect of SP or NKB on the number of DA neurons was observed. NKA treatment increased the number of DA cells as well, but at much higher concentrations (100–1000 nM; Fig. 1, B and E).
Synthetic Peptide Agonists of NK1 and NK3 but Not NK2 Receptors Mimic the Effects of Native Tachykinins. SP, NKA, and NKB are believed to act preferentially through NK1, NK2, and NK3 receptors, respectively (Pennefather et al., 2004). To determine whether selective agonists of these receptors could reproduce the effects of native tachykinins on DA neurons, we treated mesencephalic cultures with the NK1 agonist GR73632, the NK2 agonist GR64349, and the NK3 agonist [pro7]-NKB. Consistent with the results obtained with SP and NKB, the number of DA neurons increased in the presence of GR73632 and [pro7]-NKB (Fig. 1D), in a narrow range of concentrations. The peak effect was estimated at 10 nM for GR73632 and at 1 nM for [pro7]-NKB (Fig. 1D). We were surprised to find, however, that the NK2 receptor agonist GR64349 did not affect the number of TH+ cells, indicating that the action of NKA was probably mediated by its nonpreferential receptors.
Selective Antagonists of NK1 and NK3 Receptors Prevent the Effects of Tachykinins on DA Neurons. We wished first to address the specificity of the interaction between synthetic tachykinin agonists and NK receptors. Therefore, specific antagonists of NK1, NK2, and NK3 receptors (i.e., GR71251, R396, and SB218795, respectively) were added (all at 1 μM) to mesencephalic cultures in the presence of the NK1 receptor agonist GR73632 or the NK3 receptor agonist [pro7]-NKB (Fig. 2A). As expected, the NK1 receptor antagonist GR71251 prevented GR73632-mediated increase in DA cells, whereas the NK2 antagonist R396 and the NK3 antagonist SB218795 did not (Fig. 2A). Likewise, the effect of the selective NK3 agonist [pro7]-NKB on DA neurons was abolished by the corresponding antagonist SB218795 but not by the antagonists of NK1 or NK2 receptors (Fig. 2A). Note that the number of DA neurons was not further increased when we combined the NK1 and NK3 agonists at optimal concentrations (Fig. 2A).
We then intended to determine what receptor mediated the effects of native tachykinins (Fig. 2, B–D). The NK1 antagonist GR71251 was sufficient in itself to suppress the effect of SP, indicating that this tachykinin increased the number of DA neurons only through its preferential receptor (Fig. 2B). It was surprising, however, that both NK3 and NK1 antagonists were required to completely suppress the effect of NKB. Each of these antagonists alone had only partial effects (Fig. 2D). This indicates that the effect of NKB was mediated by the concomitant activation of NK1 and NK3 receptors. The effects of NKA that acts preferentially through NK2 receptors, were resistant to the selective NK2 receptor antagonist R396 (Fig. 2C), suggesting the probable involvement of nonpreferential NK receptors. Likewise, the effect of NKA was prevented by a treatment combining NK1 and NK3 receptor antagonists. Consistent with these different results, mRNA transcripts for NK1 and NK3 receptors were detected in mesencephalic cultures using RT-PCR amplification (Fig. 2E), whereas mRNA transcripts for NK2 receptors were not. Note that all three receptors were amplified from rat uterus mRNA used as a positive control (Pinto et al., 1999). Because the three native tachykinins SP, NKA, and NKB exerted their effects on DA cells through NK1 or NK3 receptors, subsequent experiments, except when indicated, were performed using the two selective NK1 and NK3 receptor agonists GR73632 and [pro7]-NKB.
The Increase in TH+ Cells Numbers after Stimulation of NK1 and NK3 Receptors Is Caused by Neuroprotection. From previous studies, we know that this culture model is characterized by a spontaneous and progressive loss of DA neurons (Michel and Agid, 1996; Salthun-Lassalle et al., 2004). Therefore, we found that 50% of TH+ neurons had disappeared by 5 DIV and more than 70% at 10 DIV (Fig. 3A). We demonstrate here that the pancaspase inhibitor Boc-D-FMK prevented DA cell loss efficiently (Fig. 3A), which confirms indirectly that a slowly occurring apoptotic process was affecting these neurons (Michel and Agid, 1996; Salthun-Lassalle et al., 2004). A long-term treatment with GR73632 (10 nM) or [pro7]-NKB (1 nM) also reduced TH+ cell loss substantially, indicating that tachykinins were probably acting by preventing the apoptotic mechanism as well. However, to exclude the possibility that NK receptor activation increased the number of TH+ neurons by stimulating cell division, we treated the cultures with the NK1 receptor agonist GR73632 in the presence of [methyl-3H]thymidine. Nuclei positive for the tritiated label were never found in association with TH+ neurons, indicating that NK1 receptor stimulation did not induce the proliferation of DA neuroblasts or their precursor cells (Fig. 3B). Note that similar results were obtained with [pro7]-NKB (data not shown).
Tachykinin-Dependent Survival of DA Neurons Is a Function of the Duration of Treatment. To determine whether shorter treatments also prevented the death of TH+ neurons, the addition of the NK3 receptor agonist [pro7]-NKB (1 nM) was delayed after plating. Under these conditions, the number of TH+ neurons that could be rescued by the treatment diminished progressively (Fig. 4A). Cultures treated continuously for 10 days (0–10 DIV) with 1 nM [pro7]-NKB had 120% more TH+ neurons than untreated cultures, whereas cultures only treated from 4 to 10 DIV had only 40% more. On the other hand, the protective effect of [pro7]-NKB was rapidly reversible if the treatment was stopped prematurely, and the cultures maintained in control medium up to 10 DIV (Fig. 4B). If [pro7]-NKB was withdrawn at day 8, only 40% of the TH+ were rescued 2 days later; it was more remarkable that, if withdrawn at 6 DIV, [pro7]-NKB produced no net increase in the number of TH+ neurons at 10 DIV (Fig. 4B). Similar results were obtained with the NK1 agonist GR73632 (data not shown).
TH+ Neurons Rescued by Activation of NK1 and NK3 Receptors Possess a Functional DA Transporter. To assess the function of the rescued DA neurons, we quantified the uptake of [3H]DA and combined microautoradiographic detection of the tritiated neurotransmitter with immunofluorescent labeling of TH. Treatments with either the NK1 agonist GR73632 or the NK3 agonist [pro7]-NKB increased the number of TH+ neurons and the number of cells accumulating [3H]DA, in the same proportion (Figs. 5A and 6). Under these conditions, virtually all TH+ neurons contained the tritiated label (Fig. 6), which indicates that both peptide agonists saved a population of neurons that was equipped with a functional DA transporter. It is interesting that the neurites of TH+ neurons treated with GR73632 and [pro7]-NKB were more intensely labeled with [3H]DA than in control cultures, suggesting that the uptake was also more efficient in these neurons. Confirming this observation, the accumulation rate of [3H]DA per TH+ neuron was augmented by approximately 2-fold in the presence of the test compounds compared with untreated cultures (Fig. 5B). In contrast, the agonist of voltage-gated Na+ channels, veratridine, which also provided robust neuroprotection to DA neurons in this model system (Salthun-Lassalle et al., 2004), had no effect on the accumulation rate of DA by these neurons (Figs. 5B and 6). Note the virtual absence of cells accumulating [3H]DA in cultures treated with the inhibitor of the DA transporter GBR-12909 (0.5 μM) before the uptake (Fig. 6).
Trophic Effects Mediated by NK1 and NK3 Receptors Are Selective for DA Neurons in Mesencephalic Cultures. TH+ cells represent approximately 1 to 2% of all neurons at the time of plating. To determine whether NK1 (GR73632) and NK3 ([pro7]-NKB) receptor agonists also affected the survival of non-DA neurons, which are predominantly GABAergic and to a lesser extent serotoninergic, we labeled the entire population of mesencephalic neurons with an antibody against MAP2. The number of MAP2+ neurons remained unchanged after long-term treatment with GR73632 or [pro7]-NKB (Fig. 7A), as illustrated in Fig. 7B. Similar results were obtained when the uptake of tritiated GABA or 5-HT was used to assess the function of GABAergic and serotoninergic neurons, respectively (Fig. 7C).
Survival Promotion by Stimulation of NK1 and NK3 Receptors Does Not Result from an Antiproliferative Effect on Glial Cells. DA neurons can be rescued efficiently in this culture model by reducing the proliferation of astrocytes or their precursor cells (Mourlevat et al., 2003). To determine whether stimulation of NK1 and NK3 receptors affected the proliferation of glial cells, we assessed the incorporation of [methyl-3H]thymidine in cultures treated continually for 7 DIV with GR73632 or [pro7]-NKB (Fig. 8). Neither GR73632 nor [pro7]-NKB had an influence on the number of thymidine+ nuclei in mesencephalic cultures (Fig. 8, B and C), which indicates that NK1 and NK3 agonists promoted the survival of DA neurons despite the glial proliferation. As expected, however, the synthetic deoxynucleoside cytosine (araC, 1 μM) caused an almost complete loss of [methyl-3H]thymidine nuclei and a robust increase in the number of TH+ neurons (Fig. 8).
The Rescue of DA Neurons Caused By Activation of NK Receptors Does Not Depend on the Secretion of a Trophic Factor. We wished to determine whether the effect mediated by NK1 and NK3 receptors was possibly mediated by GDNF, a prototypical trophic factor for DA neurons. Therefore, we tested the effects of GR73632 (10 nM) and [pro7]-NKB (1 nM) in the presence of an anti-GDNF antibody (AB 212-NA; 5 μg/ml) that neutralizes the biological activity of the trophic peptide (Fig. 9A). Whereas the antibody blocked the increase in DA cell survival produced by GDNF (10 ng/ml), it had no effect on neuronal survival promoted by tachykinins. This indicates that the effect of tachykinins was not dependent on the secretion of GDNF in the culture medium.
To exclude the effect of another putative factor secreted by glial or neuronal cells, we assessed the survival of TH+ cells maintained in a culture medium that had been conditioned previously by mesencephalic cultures exposed continually to GR73632 (10 nM) or [pro7]-NKB (1 nM). The two conditioned media protected the DA neurons but only because they contained GR73632 or [pro7]-NKB, the effects of which were blocked by the corresponding NK1 and NK3 receptor antagonists GR71251 and SB218795 (Fig. 9B).
Neuroprotection of DA Neurons by Tachykinins: Role of Voltage-Gated Ionic Channels. We have shown previously that voltage-gated ionic channels are crucially involved in the survival of DA neurons in this model system (Douhou et al., 2001; Salthun-Lassalle et al., 2004). In particular, we showed that DA neurons can be rescued from death by low-level activation of voltage-gated Na+ channels using the alkaloid veratridine (Salthun-Lassalle et al., 2004), the effects of which were blocked by the Na+ channel antagonist TTX. The effects of the agonists of NK1 (GR73632) and NK3 ([pro7]-NKB) receptors on DA neurons were prevented by TTX (100 nM), indicating that Na+ channels were also implicated in the effects of tachykinins (Fig. 10, A and C). This finding and the fact that suboptimal amounts of veratridine (0.2 μM) were able to improve the protective effect of optimal concentrations of [pro7]-NKB (Fig. 10A) and GR73632 (data not shown) suggested that the alkaloid and tachykinins promoted DA cell survival by a mechanism that was common. This was apparently not the case, because the T-type calcium-channel blocker flunarizine (5 μM), which prevented the survival-promoting effect of the alkaloid (Salthun-Lassalle et al., 2004), was unable to inhibit the effects of [pro7]-NKB or GR73632 (Fig. 10B). The neuroprotective effects of GR73632 and [pro7]-NKB were also resistant to nimodipine (10 μM), an inhibitor of the L-type calcium channels. They were reduced, however, by the snail toxin -conotoxin MVIIA (0.5 μM; Fig. 10, B and C), indicating that inward calcium currents through N-type calcium channels probably played a key role in the survival of DA neurons mediated by NK1 and NK3 receptors. As expected, the protective effects of native tachykinins SP and NKB were also blocked by treatment with TTX and -conotoxin MVIIA (Fig. 10, A and B). It is interesting that the rescuing effect of veratridine, which was prevented by TTX, remained unaffected by -conotoxin MVIIA (Fig. 10B), confirming that veratridine and tachykinins acted by mechanisms that were partly related but not identical.
To confirm that tachykinin-mediated neuroprotection was linked to activation of inward Ca2+ currents, we measured intracellular calcium levels in the presence of the various test treatments (Fig. 10D). At concentrations of NK1 and NK3 agonists that promote optimal survival of DA neurons, we observed an increase in [Ca2+]i of 60 to 80% greater than control levels. The capacity of tachykinins to increase [Ca2+]i was abolished by TTX (0.1 μM) or -conotoxin MVIIA (0.5 μM), which indicates that voltage-gated Na+ channels and N-type calcium channels played a key role in this effect (Fig. 10D). Unlike -conotoxin MVIIA, however, the L-type calcium-channel antagonist nimodipine (10 μM) and the T-type calcium-channel antagonist flunarizine (5 μM) failed to reduce the increase in [Ca2+]i induced by tachykinins (data not shown). Note that veratridine-mediated increase in intracellular calcium was blocked by TTX as expected but not by -conotoxin MVIIA (Fig. 10D).
Discussion
We demonstrate here that stimulation of NK1 and NK3 but not NK2 receptors by native tachykinins or synthetic peptide agonists provides robust and selective neuroprotection against DA cell death in mesencephalic cultures. Neuroprotection was mimicked by but independent of GDNF. It resulted from a depolarizing effect mediated through the activation of TTX-sensitive sodium channels and N-type voltage-gated calcium channels.
Native and Synthetic Tachykinins Increase the Number of DA Neurons through Activation of NK1 and NK3 Receptors. We observed that the native tachykinins SP, NKA, and NKB substantially increased the number of TH+ neurons when applied continually to mesencephalic cultures. SP, NKA, and NKB are believed to act preferentially at the NK1, NK2, and NK3 receptor sites, respectively. We wished to determine precisely, however, which of these receptors was responsible for the effects of the native tachykinins on DA neurons. The specific NK1 receptor antagonist GR71251 suppressed entirely the effect of SP on DA neurons, whereas a cotreatment combining GR71251 and the specific NK3 receptor antagonist SB218795 was required to prevent the action of NKB. This suggests, therefore, that the effect of SP occurred via NK1 receptors whereas that of NKB required activation of both NK1 and NK3 receptors. The effect of NKA seemed to be mediated entirely by its nonpreferential receptors because its action on DA neurons was resistant to the selective NK2 receptor antagonist R396 and was suppressed by a treatment combining the NK1 and NK3 receptor antagonists. Confirming that NK1 and NK3 receptors were crucial for the effects of tachykinins on DA neurons, the selective NK1 agonist GR73632 and the selective NK3 agonist [pro7]-NKB increased the number of DA neurons, whereas the NK2 agonist GR64349 had no effect. Therefore,only mRNA transcripts encoding the NK1 and NK3 receptors were detected in mesencephalic cultures.
The Activation of NK1 and NK3 Receptors Is Truly Neuroprotective for DA Neurons. The increase in the number of TH+ neurons produced by stimulation of NK1 or NK3 receptors may result from several mechanisms. The treatments may induce the proliferation of putative DA precursor cells, but this is unlikely because all TH+ neuroblasts have exited the cell cycle in the gestational day 15.5 rat mesencephalon used to generate the cultures (Rothman et al., 1980). Furthermore, the number of TH+ cells in treated cultures never exceeded that of TH+ neuroblasts detected just after plating, and [methyl-3H]thymidine, a marker of DNA synthesis, was never associated with TH+ cells in tachykinin-treated cultures. The effect of the tachykinin agonists might be simply to restore the expression of TH within neurons that contain undetectable amounts of the enzyme as they entered a premorbid state (Hirsch et al., 1988; Michel and Agid, 1996). Even though tachykinins were reported to stimulate TH protein expression (Friedman et al., 1988), this possibility is doubtful, because the tachykinin agonists failed to rescue the fraction of TH+ neurons that had already disappeared when initiation of the treatments was delayed. The observation that the pancaspase inhibitor Boc-D-FMK was also able to increase the number of DA neurons in this model system indicates that the tachykinins probably reversed an ongoing apoptotic process that is occurring spontaneously (Salthun-Lassalle et al., 2004). The underlying mechanism, however, was not related to that described for the neurotransmitter adenosine and certain antimitotics, which were effective in this culture model through the repression of glial cell proliferation (Michel et al., 1999). Finally, it is worth noting that DA neurons treated with NK1 and NK3 receptor agonists efficiently accumulated DA by active transport, which indicates that these neurons were perfectly functional when protected by tachykinins.
TTX-Sensitive Sodium Channels and N-Type Voltage-Gated Calcium Channels Are Implicated in Tachykinin-Mediated Neuroprotection. Two observations indicate that inward sodium currents were critical for tachykinin-mediated neuroprotection. The effect of NK1 and NK3 receptor agonists was mimicked by veratridine, an agonist of the voltage-gated Na+ channels (Salthun-Lassalle et al., 2004) and prevented by TTX, an irreversible antagonist of these channels (Catterall, 1980). This result is consistent with electrophysiological data showing that stimulation of NK3 receptors evokes excitatory responses in DA neurons of the substantia nigra (Nalivaiko et al., 1997). We have established previously that low-level activation of voltage-gated Na+ channels by the alkaloid veratridine protects DA neurons via a mechanism that requires calcium influx through low-threshold activated T-type calcium channels (Salthun-Lassalle et al., 2004). T-type calcium channels are reported to be activated in response to SP in certain types of neurons (Ikeda et al., 2003). In our study, however, the effects of the tachykinin agonists were resistant to flunarizine, an inhibitor of these channels (Santi et al., 2002). High-threshold-activated L- and N-type calcium channels that have been shown to contribute to the effects of tachykinins in several model systems (Bayguinov et al., 2003; Sculptoreanu and de Groat, 2003) might also participate to tachykinin-mediated neuroprotection in mesencephalic cultures. L-type calcium currents are implicated in the rescue of mesencephalic DA neurons by depolarizing concentrations of K+ (Douhou et al., 2001), but a selective antagonist of these channels, nimodipine, failed to inhibit the action of tachykinins on these neurons. In contrast, N-type calcium channels were probably essential for the survival-promoting effect of tachykinins. Indeed, blockade of these channels by the snail toxin -conotoxin MVIIA (Hirata et al., 1997) abolished neuroprotection by tachykinins. Consistent with this observation, the moderate increase in [Ca2+]i elicited by tachykinins was also reversed by -conotoxin MVIIA. This elevation was also prevented by blocking Na+ channels with TTX, which provides further support for the idea that intracellular calcium levels were crucially involved in the survival of these neurons. Finally, the efficacy of TTX to prevent the increase in calcium produced by tachykinins is an indication that Na+ inward currents may precede the activation of N-type Ca2+ channels.
The present results demonstrate that N-type calcium channels possess in common with L- and T-type calcium channels (Douhou et al., 2001; Salthun-Lassalle et al., 2004) the ability to modulate DA cell survival. These channels have different voltage ranges and rates of activation and inactivation (Hammond, 2001), which probably explains why their recruitment depends largely on the nature and intensity of the excitatory stimuli applied on DA neurons. Overall, the crucial need of DA neurons for electrical stimulation suggests that they lack adequate excitatory inputs in culture and that tachykinins could act as substitutes. It is interesting to note that only DA neurons in this model system seem dependent on such input. Finally, the rapid reversal of the effects of NK1 and NK3 receptor agonists when they are withdrawn indicates that tachykinin-dependent excitatory signals were a permanent requirement for long-term survival of DA neurons.
Is There a Link between the Survival Promotion Mediated by Tachykinins and GDNF We wished to determine whether NK1 and NK3 receptor agonists could operate through a mechanism involving GDNF, a major neurotrophic factor for DA neurons (Choi-Lundberg et al., 1997). This was excluded because a neutralizing antibody that prevented the rescue of DA neurons by GDNF did not interfere with the effects of the NK1 or NK3 receptor agonists. Other autocrine or paracrine trophic factors were probably not implicated either, as shown by our experiments with conditioned media. These experiments support the idea that tachykinins and synthetic peptide agonists acted directly on DA neurons, which express both NK1 (Futami et al., 1998) and NK3 receptors (Chen et al., 1998; Friedman et al., 2002). Doubt remains, however, because the presence of NK1 receptors on DA neurons is still a question of debate (Futami et al., 1998), and the experiments using conditioned culture media might have failed to detect molecules having a short-range diffusion or a limited lifespan. Finally, because GDNF is known to stimulate the synthesis of substance P (Ogun-Muyiwa et al., 1999), particularly in the striatum (Humpel et al., 1996), we envisaged the possibility that some of its effects might be mediated by tachykinins. This was unlikely because TTX, which prevented the rescuing effect of tachykinins on DA neurons, failed to reduce that of GDNF on the same neurons (Salthun-Lassalle et al., 2004). Therefore, as expected, the antagonists of NK1 and NK3 receptors failed to reduce the survival-promoting activity of GDNF on DA neurons as well (data not shown).
In conclusion, we have shown that stimulation of the NK1 and NK3 receptors by native or synthetic agonists exerts neuroprotective effects that are highly selective for DA neurons in mesencephalic cultures. This indicates that these neurons may depend in part on tachykinins for their survival during development. It is interesting that survival promotion mediated by tachykinins in mesencephalic cultures was mimicked by GDNF, a trophic factor that prevents the death of DA neurons during development (Oo et al., 2003). Because GDNF is still effective when applied exogenously to injured DA neurons in the adult brain (Choi-Lundberg et al., 1997; Dauer and Przedborski, 2003), it is reasonable to ask whether tachykinins could be neuroprotective in neurodegenerative conditions in which DA neurons die selectively, such as Parkinson's disease and related disorders.
Acknowledgements
We are grateful to Merle Ruberg for helpful suggestions on the manuscript.
Footnotes
This work was supported by Institut National de la Santé et de la Recherche Médicale. B.S.-L. was supported by a Doctoral Research Fellowship from Ministère de l'Education Nationale, de la Recherche et de la Technologie, and P.P.M. was supported by Pierre Fabre Research Center (Castres, France).
ABBREVIATIONS: araC, cytosine arabinoside; Boc-D-FMK, N-tert-butoxycarbonyl-D-fluoromethylketone; DA, dopamine or dopaminergic; DIV, day in vitro; GDNF, glial cell line-derived neurotrophic factor; 5-HT, serotonin; MAP2, microtubule-associated protein-2; NK, neurokinin; PBS, phosphate-buffered saline; SP, substance P; TH, tyrosine hydroxylase; TTX, tetrodotoxin; RT-PCR, reverse-transcriptase polymerase chain reaction; AB, antibody; GR71251, [D-Pro9,[spiro--lactam]Leu10,Trp11]substance P; SB218795, (R)-N-[-(methoxycarbonyl)benzyl]-2-phenylquinoline-4-carboxamide; GR73632, D-Ala-[L-Pro9,Me-Leu8]substance P(7–11); GR64349, (Lys3,Gly8-R--lactam-Leu9)neurokinin A(3–10); R396, acetyl-Leu-Asp-Gln-Trp-Phe-GlyNH2; GBR-12909, 1-(2(bis(4-fluorophenyl)methoxy)ethyl)-4-(3-phenylpropyl)piperazine di-HCl.
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