Expression and function of calcitonin gene-related peptide (CGRP) receptors in trigeminal ganglia of R192Q Cacna1a knock-in mice

Neuroscience Letters 620 (2016) 104–110

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Neuroscience Letters journal homepage: www.elsevier.com/locate/neulet

Research paper

Expression and function of calcitonin gene-related peptide (CGRP) receptors in trigeminal ganglia of R192Q Cacna1a knock-in mice Sandra Vilotti a,∗ , Natascha Vana a,1 , Arn M.J.M. Van den Maagdenberg b,c , Andrea Nistri a,∗∗ a

Neuroscience Department, International School for Advanced Studies (SISSA), Trieste, Italy Department of Neurology, Leiden University Medical Centre, Leiden, Netherlands c Department of Human Genetics, University Medical Centre, Leiden, Netherlands b

h i g h l i g h t s • • • •

CGRP is a mediator of migraine acute headache. CGRP receptors are strongly expressed by mouse trigeminal sensory neurons. In a mouse genetic model of hemiplegic migraine CGRP receptors are unchanged. Higher CGRP release instead of receptor upregulation sensitizes trigeminal ganglia.

a r t i c l e

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Article history: Received 22 February 2016 Received in revised form 22 March 2016 Accepted 24 March 2016 Available online 25 March 2016 Keywords: CGRP receptor RAMP1 CLR Familial hemiplegic migraine type-1 cAMP Nociception

a b s t r a c t Migraine is a neurovascular brain disorder suggested to be due to dysfunction of the trigeminovascular system with sensitization of trigeminal ganglion (TG) nociceptors. Since the neuropeptide calcitonin gene-related peptide (CGRP) has been established as a key player in the pathogenesis of migraine, CGRP receptor antagonists have been considered useful compounds to block headache originating from hyperactivation of such TG neurons. Whereas there is some information on the expression of CGRP receptors in postmortem human tissue, data are lacking for migraineurs suffering from common or genetic migraine. To help to clarify these issues it is very useful to study a transgenic knock-in (KI) mouse model of hemiplegic migraine expressing a R192Q missense mutation in the ␣1 subunit of CaV 2.1 calcium channels previously found in patients with familial hemiplegic migraine type-1 (FHM-1). The aim of the present study, therefore, was to compare CGRP receptor expression and function in wildtype (WT) versus KI mouse TG. The principal components of the CGRP receptor, namely the CLR and RAMP-1 proteins, were similarly expressed in WT and KI TG neurons (in situ or in culture) and responded to exogenous CGRP with a strong rise in cAMP concentration. Hence, the previously reported phenotype of sensitization of KI TG neurons is not due to up-regulation of CGRP receptors but is likely caused by a constitutively larger release of CGRP. This observation implies that, in FHM-1 TG, normal TG sensory neuron signaling can be restored once the extracellular concentration of CGRP returns to control level with targeted treatment. © 2016 Elsevier Ireland Ltd. All rights reserved.

1. Introduction

Abbreviations: CLR, calcitonin receptor-like receptor; CGRP, calcitonin generelated peptide; FHM-1, familial hemiplegic migraine type-1; RAMP1, receptor activity-modifying protein 1; s.d., standard deviation; TG, trigeminal ganglion. ∗ Corresponding author at: SISSA, Via Bonomea 265, 34136 Trieste, Italy. ∗∗ Corresponding author. E-mail addresses: [email protected] (S. Vilotti), [email protected] (N. Vana), A.M.J.M.van den [email protected] (A.M.J.M. Van den Maagdenberg), [email protected] (A. Nistri). 1 Present address: Centre for Biological Sciences, Life Sciences Building 85, University of Southampton, Southampton SO17 1BJ. http://dx.doi.org/10.1016/j.neulet.2016.03.046 0304-3940/© 2016 Elsevier Ireland Ltd. All rights reserved.

Migraine is a complex, painful and debilitating neurovascular brain disorder proposed to involve dysfunction of the trigeminovascular system [1]. While the etiology of migraine is largely unknown, it has long been recognized that the 37 amino acid neuropeptide calcitonin gene-related peptide (CGRP) is involved in its pathophysiology [2]. CGRP receptor antagonists have shown efficacy in the treatment of migraine attacks [3,4]. Under normal physiological conditions CGRP receptors are expressed throughout the trigeminovascular system, including cranial vessels [5–7], second-order brainstem sensory neurons [6,8], and trigeminal

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sensory ganglia [6,9]. Expression of CGRP receptors has been examined in human, monkey and rat trigeminal ganglia (TG) [6,9] where CGRP receptor immunoreactivity has been found in about 40% of neurons and in some non-neuronal cells. This is particularly interesting because recent studies pointed out that TG neurons may be a potential therapeutic site of action for CGRP receptor antagonists [10]. Nonetheless, there is so far a paucity of data concerning the expression of CGRP receptors in migraineurs or in animal models of migraine. To generate functional responses, CGRP receptors require the seven transmembrane calcitonin-like receptor (CLR), which forms the ligand binding site for CGRP, plus an accessory protein termed receptor activity-modifying protein-1 (RAMP1; [11]). A typical effect of CGRP receptor activation is a rise in intracellular cAMP synthesis [11,12]. Despite the rich complement of receptor signaling at the level of the trigeminal territory, experimental application of CGRP is very slow to change firing activity by TG neurons [13], outlining the possibility that the action of CGRP might be indirect via non-neuronal cells and mediated through release of other mediators [13,14]. Because of the usefulness of genetic mouse models to investigate basic mechanisms of migraine pathophysiology [15], the present report studied the expression of CGRP receptors in the mouse TG in situ and in culture. In particular, we compared CGRP receptor expression and function in TG of WT and a transgenic knock-in (KI) mouse model of hemiplegic migraine that expresses the R192Q missense mutation in the ␣1 subunit of CaV 2.1 calcium channels known to cause familial hemiplegic migraine type-1 (FHM-1) in patients [16,17]. In previous studies, TG cultures from mutant KI mice were shown to release more CGRP [18], likely generating neuronal sensitization [19]. The present study, therefore, addressed the issue whether such constitutively larger CGRP release from mutant KI TG neurons is accompanied by deranged expression of CGRP receptors. 2. Materials and methods 2.1. Animals and TG primary culture preparations Homozygous CaV 2.1 R192QKI and WT littermates [16], and C57BL/6J mice were used for the study. All animal procedures were conducted in accordance with guidelines of the Italian Animal Welfare Act and regulations of animal welfare and approved by the Scuola Internazionale Superiore di Studi Avanzati (SISSA) ethics committee. Genotyping was performed by PCR as previously reported [16]. TG primary cultures were obtained from animals at the age of P12-14 following general anesthesia with slowly raising levels of CO2 [20] and used 24 or 48 h after plating. Ganglion tissue samples and cultures were collected and processed in parallel for WT and R192Q KI mice. 2.2. Western Blot Western blot analysis was performed according to methods previously reported [21]. Cells were lysed in buffer solution (50 mM Tris-HCl pH 7.5, 150 mM NaCl, 2%, 0,1% Nonidet P-40, 0,1% sodium deoxycholate, 0,1% SDS, 2 mM EDTA plus protease inhibitors mixture; Complete, Roche Applied Science, Basel, Switzerland) and immunoblotted with the following primary antibodies: antiRAMP1 (1:1000; goat 844, Merck and Co., Inc, USA) and anti-CLR (1:1000; rabbit 3155, Merck and Co.), anti-␤-actin (1:5000; A5441, Sigma Milan, Italy), anti-␤-tubulin III (1:3000; T5076, Sigma). Signals were revealed after incubation with secondary antibodies conjugated with horseradish peroxidase by using the ECL detection system (Amersham Biosciences, Piscataway, NJ, USA) and recorded

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by the Alliance 4.7 (UVITEC, Cambridge, UK) digital imaging and normalized with respect to the levels of ␤-tubulin III or ␤-actin used as gel loading control. 2.3. Preparations of mouse trigeminal ganglia and immunostaining For immunocytochemistry, primary cultures of P12 mouse trigeminal ganglia were prepared as described previously [22] and used 24 h after plating. For in situ tissue immunohistochemistry, mice were deeply anesthetized with intraperitoneal injection of urethane (0.3 mL of 1 g/mL; Sigma) and perfused transcardially with phosphate buffer solution followed by 4% paraformaldehyde. Trigeminal ganglia were removed, and processed as previously reported [21]. Each experiment was performed on an average of 5 cryostat-cut serial longitudinal slices (12 ␮m-thick) sampled every ∼70 ␮m, and thus covering the entire ganglion. Antibodies against RAMP1 (1:100; Merck and Co. goat 844), CLR (1:500; Merck and Co. rabbit 3155), and ␤-tubulin III (1:1000; Sigma, mouse T5076) were used. Secondary antibodies conjugated with Alexa Fluor-488 or Alexa Fluor-594 (1:500; Invitrogen, Milan, Italy, goat anti-mouse and anti-rabbit) were then employed. Nuclei were counterstained with DAPI (Sigma). Images from whole ganglion sections and from cell cultures were visualized with Leica confocal microscope (Leica TCS SP2, Wetzlar, Germany) or a Zeiss Axioskop fluorescence microscope (Zurich, Switzerland). Similar procedures were used for fluorescence immunostaining of cultured mouse TG neurons. The specificity of the RAMP1 and CLR antibodies used in this study has been previously validated [8,22]. 2.4. Controls and blocking peptides To verify the specificity of the immunohistochemical reaction, every staining was controlled by omitting primary antibodies in the incubation fluid. Pre-absorption controls were performed with all CLR and RAMP1 primary antibodies using specific blocking peptides (ratio 100:1 peptide/antibody concentration) supplied by the same antibody manufacturers. The blocking peptides were re-suspended in sterile water and then incubated in PBST containing 1% BSA and 3% normal goat serum, with primary antibodies, at 4◦ C. Sections incubated with antibodies alone versus blocked antibodies were compared. 2.5. ELISA assay Basal and CGRP-evoked intracellular cAMP concentrations were measured in primary TG cultures, 24 h after plating, with a commercial cAMP ELISA kit (Abnova, Taoyuan City, Taiwan) following manufacturer’s instructions. In each well, homogenates from two TGs were loaded (in duplicate); the global data were from three independent experiments. Data were normalized to total protein concentrations of cell lysates as determined with the bicinchoninic acid method (Sigma). The cAMP concentrations were extrapolated from a best-fit line calculated from serial dilutions of a cAMP sample standard, and were then expressed (pmol/␮g protein) as change versus untreated control. 2.6. Statistical analysis Data are expressed as mean ± standard deviation (s.d.), where n indicates the number of independent experiments or the number of investigated cells, as indicated in figure legends. Statistical analysis was performed using nonparametric Mann-Whitney rank sum test, or the Student’s t-test, after the software directed choice of non-parametric or parametric data, respectively (Matlab; Sigma

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Fig. 1. Western blot analysis of mouse CGRP receptors expressed in mouse TG ex vivo and in vitro. Representative example of Western immunoblotting showing RAMP1 (left) or CLR (right) in whole TG lysate from P30 mice (A) or from primary TG culture (B), (lane 2 in each panel). Lysates of HEK293T cells (lane 1) were used as negative control for evaluating antibody specificity.

Plot and Sigma Stat Software, Chicago, IL, USA). A p-value of <0.05 was accepted as indicative of a statistically significant difference.

3. Results 3.1. Specificity of CLR and RAMP1 antibodies In view of the debated specificity of antibodies that label CGRP receptors, it seemed important to use antibodies against CLR and RAMP1 from the same source as those previously used and validated to characterize the expression of CGRP receptors in rat and human TG [9,23]. To this end, Western blotting was performed to first test the specificity of the antibodies and their cross-reactivity for mouse preparations as shown in Fig. 1. RAMP1 protein is known to exist as a monomer (∼15 kDa) and dimer (∼30 kDa), even under reducing gel electrophoresis conditions [24,25]. As depicted in Fig. 1, Western immunoblotting with antiRAMP1 antibody in whole TG lysates produced a band of ∼15 kDa (consistent with the monomer), and three bands with molecular mass of approximately 30–35 kDa (most likely dimers of RAMP1) in accordance with freely-available data provided at the following link: http://www.scbt.it/datasheet-11379-ramp1-fl-148-antibody. html. One likely possibility is that, under basal conditions, the dimer RAMP1 underwent post-translational modifications such as glycosylation. As negative control, cell lysate of HEK293 cells showed no signal, which is in accordance with the previous validation of these antibodies [9]. The antibody against CLR recognized two proteins with molecular masses of about 54 kDa and 60 kDa (Fig. 1A, right), consistent

with the predicted mass of mature and core-glycosylated CLR protein [24,26]. No significant signals were detected in control HEK293T cell lysate, confirming the specificity of the antibody. The presence of both RAMP1 and CLR proteins was also observed in cultured TG cells as demonstrated by Western blotting (Fig. 1B), where the patterns of bands were similar to those observed in whole TG lysate.

3.2. Distribution of CLR and RAMP1 in mouse TG Fig. 2A shows CLR or RAMP1 cytoplasmic immunoreactivity of neurons in mouse TG tissue in accordance with data from other animal species [6,9]. Systematic cell counting indicated that 35% of neurons (identified with ␤-tubulin staining) were CLRimmunopositive, while the number of RAMP1-positive neurons was about 37%. Besides neurons, satellite glial cells and other non-neuronal cells stained, not uncommonly, positive for CLR and RAMP1 (Fig. 2), in analogy with previous findings [6,9,23]. Double labeling with CLR and RAMP1 revealed that in the vast majority of cells co-localization of these receptor components, which is necessary to form mature CGRP receptors, did occur [11]. In fact, the percentage of neurons exclusively immunopositive to either CLR or RAMP1 was very small (2.5% CLR + /RAMP1-, and 1.5% CLR-/RAMP1 + neurons). Pre-absorption controls for CLR or RAMP1 antibodies, using their respective blocking peptide, and all other negative controls (omission of primary antibody, see Section 2) yielded no immunoreactive signal (Fig. 2B). Because of the extensive use of TG cultures to study TG nociceptive processes [4], we also looked for immunocytochemistry

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Fig. 2. Localization of CLR and RAMP1 in the mouse TG. (A) Representative confocal images of longitudinal sections of adult TG. Immunohistochemistry reveals CLR and RAMP1 immunoreactivity in neurons (long arrows) and non-neuronal cells (arrowheads). Cells negative for CLR or RAMP1 are indicated with short arrows. Cell nuclei are visualized with DAPI staining (blue). Double immunolabeling of CLR and RAMP1 in longitudinal sections showed extensive co-staining of TG neurons (see also enlarged image) and of non-neuronal cells. (B) Immunostaining with anti-CLR and anti-RAMP1 antibodies versus pre-absorption with their respective blocking peptides. No positive immunoreactivity was observed when blocking peptides were used. In negative controls (use of only secondary antibodies, Alexa 488 and Alexa 594), no immunoreactivity was observed, except for the lipofuscin auto-fluorescence. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

signals of CLR and RAMP1 in cultured mouse trigeminal ganglia (Fig. 3). Double staining with the neuron-specific ␤-tubulin (Fig. 3) enabled us to count neurons positive for CLR and/or RAMP1. Systematic cell counting indicated that 36% of ␤-tubulin-positive cells expressed CLR, consistent with our results from tissue sections.

RAMP1-immunopositive cells accounted for 37% of the total neuronal population. Staining for both proteins was stronger in the neuronal cell soma, but was also seen in their processes. Immunocytochemical staining for CLR or RAMP1 was occasionally observed in non-neuronal cells (presumed to be satellite glial cells and fibrob-

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Fig. 3. CLR and RAMP1 are expressed in mouse TG cultures. (A) Immunocytochemical analysis of CLR or RAMP1 (green) and ␤-tubulin (red) in primary TG culture from P12 mice. Nuclei are visualized with DAPI (blue). Merged image indicates extensive co-staining in ganglion neurons. Larger magnification images (right) show examples of CLR and RAMP1 immunoreactive cells. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

lasts) although the signal was consistently weaker in analogy with data from rat TG cultures [26]. Glial expression of these proteins is in agreement with the finding of CLR and functional CGRP receptors in cultured astroglia [27]. 3.3. Characterization of CGRP receptors in TG from WT and CaV 2.1 R192Q KI mice In TG tissue sections from WT or KI mice similar expression of CLR and RAMP1 was detected as exemplified in Fig. 4A (left), and quantified in Fig. 4A (right). Analogous data were obtained from primary TG cultures in which CLR receptor immunoreactivity was readily detected in 34% of KI neurons (Fig. 4B) as shown by its colocalization with ␤-tubulin, and similar to the percentage observed in WT (35%; Fig. 4B). In untreated TG cultures from WT animals, 38% of neurons were identified as RAMP1-positive, while in KI ganglion culture RAMP1-immunopositive cells were 37% (Fig. 4B). Western blot analysis did not show substantial genotypic variation of CLR or RAMP1 protein levels in TG primary cultures from young (P12) WT or KI mice (Fig. 4C). We also examined whether the CGRP receptor system was similarly functional in KI and WT TG. To this end, we compared the effect of CGRP on intracellular cAMP of WT and KI cultures. Fig. 4D shows that application of CGRP (1 ␮M; 5, 10 and 30 min) caused a similar rise in cAMP in WT and KI samples in a time- and concentrationrelated fashion. These effects were prevented by pre-application of the antagonist CGRP 8-37 (1 ␮M). Overall, these data indicated no significant genotypic difference in the expression of CGRP receptors suggesting that previously-reported increase in basal CGRP release typical of KI TG [18] is not accompanied by modulation of CGRP receptor expression in KI TG. 4. Discussion The present study demonstrates CGRP receptor expression in mouse TG neurons, their functional activity in control conditions as well as in a hemiplegic migraine mouse model [15] in which basal, high levels of the peptide are present [28]. The implications of the current findings are that the previously observed TG sensitiza-

tion by large concentrations of CGRP [18] does not elicit persistent adaptive changes in CGRP receptor expression or activity. In control tissue, one can surmise that fluctuations in extracellular CGRP caused by various stressors [29] render trigeminal ganglion cells excitable for stimuli present in the normal environment and may, thus, trigger TG firing via a slow process that needs trafficking and neosynthesis of ionotropic receptors responsible for afferent signaling to brainstem nuclei. Nevertheless, once the rise in ambient CGRP subsides, standard CGRP receptor signaling is reinstated. Conversely, in R192Q mutant mice, despite normal CGRP receptor expression, the constitutively larger CGRP concentrations confer enhanced TG firing [19] plus pain behavior consistent with trigeminal nociception [30]. This interpretation is even in keeping with a report of low amounts of CGRP in KI ganglia, which is possibly due to a higher peptide release [31]. Our cAMP assay validated the slowly developing response of WT or KI TG to CGRP that, when applied at a concentration eliciting near maximal effects [22], required at least 10 min to evoke a peak response. In view of the multiple intracellular pathways modulated by cAMP [32], it is not surprising that before CGRP-induced sensitization of TG nociceptors is manifested there is up to 1 h delay [22]. Consistent with the multistep nature of this complex process is the observation that return of TG firing to baseline requires long-lasting blockage of CGRP receptors [19]. 5. Conclusion Because the neuropeptide CGRP has a key role in migraine pathophysiology, TG neurons may be a potential therapeutic site of action for CGRP receptor antagonists. However, little is known about the expression of CGRP receptors in migraineurs. Furthermore, the delayed effect of CGRP on neuronal excitability might question whether the peptide acts directly on neuronal receptors and how many TG neurons might respond to it. The present study reveals similar functional CGRP receptor expression levels in mouse TG neurons of WT mice and transgenic mice that are a model for hemiplegic migraine, possibly even for common forms of migraine [15]. These data suggest that the formerly identified larger CGRP release from KI TG is not accompanied by any altered expression

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Fig. 4. Characterization of CLR and RAMP receptors in TG from WT and KI mice. Representative immunocytochemical examples of endogenous CLR and RAMP1 expression in WT (left panel) and KI (right panel) TG ganglia (A) or TG cultures (B). Nuclei are visualized with DAPI (blue). Note extensive co-staining of CLR (red) and RAMP1 (green). The histograms quantify percentage of CLR- and RAMP1-positive cells in WT and KI (n = 4; *p < 0.05, Kruskal-Wallis test). (C), Representative Western blot images showing protein expression of CLR and RAMP1 in TG from P30 mice. ␤-Actin was used as a loading control. Histograms (right) quantify CLR and RAMP1 relative optical density values for each examined group (n = 4). (D) Histograms quantify the fold-change of cAMP concentration evoked by CGRP (1 ␮M; 5, 10, and 15 min) in TG extracts from WT and KI TG cultures, with respect to basal conditions. The effect of pre-application of peptide antagonist CGRP 8-37 (1 ␮m) is also shown (n = 4). (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

of CGRP receptors. Thus, larger release of CGRP rather than upregulation of its receptors seem to be the predominant contributor to trigeminal pain sensitization of KI neurons.

Authors’ contributions All authors read and approved the final manuscript. NV, SV, design of experiments and collection of data; AMJMVDM, mouse genetic model supply; AN, project supervision; SV, AMJMVDM, AN, joint contribution to MS writing. The authors declare that they have no competing interests.

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