Sajedeh Eftekhari

Differential distribution of calcitonin gene-related peptide and its receptor components in the human trigeminal ganglion

Calcitonin gene related peptide (CGRP) has a key role in migraine and recently CGRP receptor antagonists have demonstrated clinical efficacy in the treatment of migraine. However, it remains unclear where the CGRP receptors are located within the CGRP signaling pathway in the human trigeminal system and hence the potential antagonist sites of action remain unknown. Therefore we designed a study to evaluate the localization of CGRP and its receptor components calcitonin receptor-like receptor (CLR) and receptor activity modifying protein (RAMP) 1 in the human trigeminal ganglion using immunohistochemistry and compare with that of rat. Antibodies against purified CLR and RAMP1 proteins were produced and characterized for this study. Trigeminal ganglia were obtained at autopsy from adult subjects and sections from rat trigeminal ganglia were used to compare the immunostaining pattern. The number of cells expressing CGRP, CLR and RAMP1, respectively, were counted. In addition, the glial cells of trigeminal ganglion, particularly the satellite glial cell, were studied to understand a possible relation. We observed immunoreactivity for CGRP, CLR and RAMP1, in the human trigeminal ganglion: 49% of the neurons expressed CGRP, 37% CLR and 36% RAMP1. Co-localization of CGRP and the receptor components was rarely found. There were no CGRP immunoreactions in the glial cells; however some of the glial cells displayed CLR and RAMP1 immunoreactivity. Similar results were observed in rat trigeminal ganglia. We report that human and rat trigeminal neurons store CGRP, CLR and RAMP1; however, CGRP and CLR/RAMP1 do not co-localize regularly but are found in separate neurons. Glial cells also contain the CGRP receptor components but not CGRP. Our results indicate, for the first time, the possibility of CGRP signaling in the human trigeminal ganglion involving both neurons and satellite glial cells. This suggests a possible site of action for the novel CGRP receptor antagonists in migraine therapy.

Calcitonin gene-related peptide (CGRP) and its receptor components in human and rat spinal trigeminal nucleus and spinal cord at C1-level

BackgroundCalcitonin gene-related peptide (CGRP) has a key role in migraine pathophysiology and is associated with activation of the trigeminovascular system. The trigeminal ganglion, storing CGRP and its receptor components, projects peripheral to the intracranial vasculature and central to regions in the brainstem with Aδ- and C-fibers; this constitutes an essential part of the pain pathways activated in migraine attacks. Therefore it is of importance to identify the regions within the brainstem that processes nociceptive information from the trigeminovascular system, such as the spinal trigeminal nucleus (STN) and the C1-level of the spinal cord. Immunohistochemistry was used to study the distribution and relation between CGRP and its receptor components - calcitonin receptor-like receptor (CLR) and receptor activity modifying protein 1 (RAMP1) - in human and rat STN and at the C1-level, using a set of newly well characterized antibodies. In addition, double-stainings with CGRP and myelin basic protein (MBP, myelin), synaptophysin (synaptic vesicles) or IB4 (C-fibers in general) were performed.ResultsIn the STN, the highest density of CGRP immunoreactive fibers were found in a network around fiber bundles in the superficial laminae. CLR and RAMP1 expression were predominately found in fibers in the spinal trigeminal tract region, with some fibers spanning into the superficial laminae. Co-localization between CGRP and its receptor components was not noted. In C1, CGRP was expressed in fibers of laminae I and II. The CGRP staining was similar in rat, except for CGRP positive neurons that were found close to the central canal. In C1, the receptor components were detected in laminae I and II, however these fibers were distinct from fibers expressing CGRP as verified by confocal microscopy.ConclusionsThis study demonstrates the detailed expression of CGRP and its receptor components within STN in the brainstem and in the spinal cord at C1-level, and shows the possibility of CGRP acting postjunctionally in these areas putatively involved in primary headaches.

Localization of CGRP, CGRP receptor, PACAP and glutamate in trigeminal ganglion. Relation to the blood-brain barrier.

Calcitonin gene-related peptide (CGRP) receptor antagonists have demonstrated anti-migraine efficacy. One remaining question is where do these blockers act? We hypothesized that the trigeminal ganglion could be one possible site. We examined the binding sites of a CGRP receptor antagonist (MK-3207) and related this to the expression of CGRP and its receptor in rhesus trigeminal ganglion. Pituitary adenylate cyclase-activating polypeptide (PACAP) and glutamate were examined and related to the CGRP system. Furthermore, we examined if the trigeminal ganglion is protected by the blood-brain barrier (BBB). Autoradiography was performed with [(3)H]MK-3207 to demonstrate receptor binding sites in rhesus trigeminal ganglion (TG). Immunofluorescence was used to correlate binding and the presence of CGRP and its receptor components, calcitonin receptor-like receptor (CLR) and receptor activity-modifying protein 1 (RAMP1), and the distribution of PACAP and glutamate in rhesus and rat TG. Evans blue was used to examine large molecule penetration into the rat TG. High receptor binding densities were found in rhesus TG. Immunofluorescence revealed expression of CGRP, CLR and RAMP1 in trigeminal cells. CGRP positive neurons expressed PACAP but not glutamate. Some neurons expressing CLR and RAMP1 co-localized with glutamate. Evans blue revealed that the TG is not protected by BBB. This study demonstrates CGRP receptor binding sites and expression of the CGRP receptor in rhesus and rat TG. The expression pattern of PACAP and glutamate suggests a possible interaction between the glutamatergic and CGRP system. In rat the TG is outside the BBB, suggesting that molecules do not need to be CNS-penetrant to block these receptors.

Possible sites of action of the new calcitonin gene-related peptide receptor antagonists.

Migraine is considered a neurovascular disease affecting more than 10% of the general population. Currently available drugs for the acute treatment of migraine are vasoconstrictors, which have limitations in their therapeutic use. The calcitonin gene-related peptide (CGRP) has a key role in migraine, where levels of CGRP are increased during acute migraine attacks. CGRP is expressed throughout the central and peripheral nervous system, consistent with control of vasodilatation and transmission of nociceptive information. In migraine, CGRP is released from the trigeminal system. At peripheral synapses CGRP results in vasodilatation via receptors on the smooth muscle cells. At central synapses, CGRP acts postjunctionally on second-order neurons to transmit pain centrally via the brainstem and midbrain to higher cortical pain regions. The recently developed CGRP-receptor antagonists have demonstrated clinical efficacy in the treatment of acute migraine attacks. A remaining question is their site of action. The CGRP-receptor components (calcitonin receptor-like receptor, receptor activity modifying protein 1 and receptor component protein) are found to colocalize in the smooth muscle cells of intracranial arteries and in large-sized neurons in the trigeminal ganglion. The CGRP receptor has also been localized within parts of the brain and the brainstem. The aim of this paper is to review recent localization studies of CGRP and its receptor components within the nervous system and to discuss whether these sites could be possible targets for the CGRP-receptor antagonists.

A second trigeminal CGRP receptor: function and expression of the AMY1 receptor

The trigeminovascular system plays a central role in migraine, a condition in need of new treatments. The neuropeptide, calcitonin gene‐related peptide (CGRP), is proposed as causative in migraine and is the subject of intensive drug discovery efforts. This study explores the expression and functionality of two CGRP receptor candidates in the sensory trigeminal system.

Differentiation of Nerve Fibers Storing CGRP and CGRP Receptors in the Peripheral Trigeminovascular System.

Background The dura mater with the meningeal artery has since long been hypothesized to play an important role in migraine. It has been suggested that neuropeptides such as calcitonin gene-related peptide (CGRP) and substance P can activate dura mast cells leading to secretion of vasoactive, pro-inflammatory and neurosensitzing mediators, thereby contributing to migraine pathogenesis. Method: Immunofluorescence was used to study the detailed distribution of and its receptor componentscalcitonin receptor-like receptor (CLR) and receptor activity modifying protein 1 (RAMP1)in whole-mount rat dura mater, using a set of newly characterized antibodies. Their relation to each other, to mast cells, myelin, substance P, neuronal nitric oxide synthase (nNOS), pituitary adenylate cyclase-activating polypeptide (PACAP) and vasoactive intestinal peptide (VIP) were studied. In addition, we examined expression of CGRP and its receptor components in fresly isolated human dura vessels.

Cerebellar distribution of calcitonin gene-related peptide (CGRP) and its receptor components calcitonin receptor-like receptor (CLR) and receptor activity modifying protein 1 (RAMP1) in rat

Clinical and experimental results have revealed a fundamental role of calcitonin gene-related peptide (CGRP) in primary headaches. CGRP is widely expressed in neurons both in the central nervous system (CNS) and in peripheral sensory nerves. In the CNS there is a wide distribution of CGRP-containing neurons with the highest levels seen in striatum, amygdale and cerebellum. Moreover, in acute attacks of migraine there is evidence of cerebellar activation. To understand the role of CGRP, antibodies towards the CGRP receptor components calcitonin receptor-like receptor (CLR) and receptor activity modifying protein type 1 (RAMP1) have been developed. In the present study we therefore examined immunohistochemically the distribution of CGRP and its receptor components in the cerebellum. CGRP immunoreactivity was only found intracellularly in the cerebellar Purkinje cell bodies, whereas CLR and RAMP1 were detected on the surface of the Purkinje cell bodies and in their processes. The elaborate dendritic tree of Purkinje cell fibers was distinctly visualized with the RAMP1 antibody. In addition, profoundly stained fibers spanning from the molecular layer into the medulla was observed with the RAMP1 antibody. Judged from the high density of immunoreactive cells expressing CGRP, RAMP1 or CLR, and from the double staining of CGRP and RAMP1 it is likely that most, if not all, Purkinje cells express both the peptide and the receptor components. Double staining with RAMP1 and the glial cell markers glial fibrillary acidic protein (GFAP) and S-100 revealed an almost identical staining pattern of the antibodies in the area of the cell body surfaces. However, as judged by confocal microscopy, no double staining was present. Instead, it was discovered that the glial cells tightly surrounded the Purkinje cells which easily could be interpreted as co-localization in the epifluorescence microscope. Our observations demonstrate that there is a rich expression of CGRP and CGRP receptor elements in the cerebellum which points towards a functional role of CGRP in cerebellar Purkinje cells. Recent advances in the biology of the cerebellum indicate that there may be a role in nociception; hence a target of the recently discovered CGRP receptor antagonists that have demonstrated improvement in migraine pain and associated symptoms could be cerebellar CGRP receptors.

Effect of the calcitonin gene-related peptide (CGRP) receptor antagonist telcagepant in human cranial arteries.

Introduction: Calcitonin gene-related peptide (CGRP) is a neuronal messenger in intracranial sensory nerves and is considered to play a significant role in migraine pathophysiology. Materials and methods: We investigated the effect of the CGRP receptor antagonist, telcagepant, on CGRP-induced cranial vasodilatation in human isolated cerebral and middle meningeal arteries. We also studied the expression of the CGRP receptor components in cranial arteries with immunocytochemistry. Concentration response curves to αCGRP were performed in human isolated cerebral and middle meningeal arteries in the absence or presence of telcagepant. Arterial slices were stained for RAMP1, CLR and actin in a double immunofluorescence staining. Results: In both arteries, we found that: (i) telcagepant was devoid of any contractile or relaxant effects per se; (ii) pretreatment with telcagepant antagonised the αCGRP-induced relaxation in a competitive manner; and (iii) immunohistochemistry revealed expression and co-localisation of CLR and RAMP1 in the smooth muscle cells in the media layer of both arteries. Conclusions: Our findings provide morphological and functional data on the presence of CGRP receptors in cerebral and meningeal arteries, which illustrates a possible site of action of telcagepant in the treatment of migraine.

Characterization of the Calcitonin Gene-Related Peptide Receptor Antagonist Telcagepant (MK-0974) in Human Isolated Coronary Arteries

The sensory neuropeptide calcitonin gene-related peptide (CGRP) plays a role in primary headaches, and CGRP receptor antagonists are effective in migraine treatment. CGRP is a potent vasodilator, raising the possibility that antagonism of its receptor could have cardiovascular effects. We therefore investigated the effects of the antimigraine CGRP receptor antagonist telcagepant (MK-0974) [N-[(3R,6S)-6-(2,3-difluorophenyl)-2-oxo-1-(2,2,2-trifluoroethyl)azepan-3-yl]-4-(2-oxo-2,3-dihydro-1H-imidazo[4,5-b]pyridine-1-yl)piperidine-1-carboxamide] on human isolated coronary arteries. Arteries with different internal diameters were studied to assess the potential for differential effects across the coronary vascular bed. The concentration-dependent relaxation responses to human αCGRP were greater in distal coronary arteries (i.d. 600–1000 μm; Emax = 83 ± 7%) than proximal coronary arteries (i.d. 2–3 mm; Emax = 23 ± 9%), coronary arteries from explanted hearts (i.d. 3–5 mm; Emax = 11 ± 3%), and coronary arterioles (i.d. 200–300 μm; Emax = 15 ± 7%). Telcagepant alone did not induce contraction or relaxation of these coronary blood vessels. Pretreatment with telcagepant (10 nM to 1 μM) antagonized αCGRP-induced relaxation competitively in distal coronary arteries (pA2 = 8.43 ± 0.24) and proximal coronary arteries and coronary arterioles (1 μM telcagepant, giving pKB = 7.89 ± 0.13 and 7.78 ± 0.16, respectively). αCGRP significantly increased cAMP levels in distal, but not proximal, coronary arteries, and this was abolished by pretreatment with telcagepant. Immunohistochemistry revealed the expression and colocalization of the CGRP receptor elements calcitonin-like receptor and receptor activity-modifying protein 1 in the smooth muscle cells in the media layer of human coronary arteries. These findings in vitro support the cardiovascular safety of CGRP receptor antagonists and suggest that telcagepant is unlikely to induce coronary side effects under normal cardiovascular conditions.

A Battery of Cell- and Structure-specific Markers for the Adult Porcine Retina.

The pig is becoming an increasingly used non-primate model in experimental studies of human retinal diseases and disorders. The anatomy, size, and vasculature of the porcine eye and retina closely resemble their human counterparts, which allows for application of standard instrumentation and diagnostics used in the clinic. Despite many reports that demonstrate immunohistochemistry as a useful method for exploring neuropathologi-cal changes in the mammalian central nervous system, including the pig, the porcine retina has been sparsely described. Hence, to facilitate further immunohistochemical analysis of the porcine retina, we report on the successful use of a battery of antibodies for staining of paraformaldehyde-fixed cryosectioned retina. The following antibodies were evaluated for neuronal cells and structures: recoverin (cones and rods), Rho4D2 (rods), transducin-γ (cones), ROM-1 (photoreceptor outer segments), calbindin (horizontal cells), PKC-α (bipolar cells), parvalbumin (amacrine and displaced amacrine cells), and NeuN (ganglion cells and displaced amacrines). For detecting synaptic connections in fiber layers, we used an antibody against synaptobrevin. For detecting retinal pigment epithelium, we studied antibodies against cytokeratin and RPE65, respectively. The glial cell markers used were bFGF (Müller cells and displaced amacrine cells), GFAP (Müller cells and astrocytes), and vimentin (Müller cells). Each staining effect was evaluated with regard to its specificity, sensitivity, and repro-ducibility in the identification of individual cells, specific cell structures, and fiber layers, respectively. The markers parvalbumin and ROM-1 were tested here for the first time for the porcine retina. All antibodies tested resulted in specific staining of high quality. In conclusion, all immunohistochemical protocols presented here will be applicable in fixed, cryosectioned pig retina.