ISSN doi: /head VC 2017 American Headache Society Published by Wiley Periodicals, Inc.

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1 ISSN Headache doi: /head VC 2017 American Headache Society Published by Wiley Periodicals, Inc. Supplement Article The Trigeminovascular Pathway: Role of CGRP and CGRP Receptors in Migraine Lars Edvinsson, MD, PhD The trigeminal ganglion plays a key role in primary headache pathophysiology. Calcitonin gene-related peptide (CGRP) and CGRP receptors are expressed in trigeminal neurons that form C-fibers and A-fibers, respectively. In acute migraine and cluster headache attacks, there is release of CGRP into the cranial venous outflow. In addition, intravenous CGRP can induce migraine-like symptoms in migraine patients. These findings led to the development of anti-migraine therapies that inhibit CGRP action. Currently, CGRP receptor antagonists, the gepants, and monoclonal antibodies towards CGRP and the CGRP receptor are all showing positive relief of acute and chronic migraine in clinical trials. However, there is still much to learn about the role of CGRP and CGRP receptors in headache pathophysiology, the critical anatomical sites, peripheral or central, of anti-cgrp agents, and the potential involvement of CGRP-related peptides and receptors. This review provides a brief history of the discovery of the role of CGRP in migraine and highlights current progress in understanding the complexity of the trigeminovascular pathway and its peptide transmitters. Key words: CGRP, CGRP receptor, CLR, RAMP1, gepants, monoclonal antibodies (Headache 2017;57:47-55) Primary headache and migraine in particular, is among the top 10 most common causes of disability in the world, posing a heavy burden on the individual and society. 1 Promising results from experimental and clinical translational research are providing a better understanding of the underlying neurobiology of this disorder and options for improved therapy. Over the years, many neurotransmitters or neuromodulators have been considered to be involved in migraine but none were shown to be a key player. It now appears that the CGRP pathway plays a From the Department of Medicine, Lund University, Lund, Sweden. Address all correspondence to L. Edvinsson, Professor, Department of Medicine, University Hospital, Lund, Sweden. Accepted for publication March 7, dominant role in migraine; this was first suggested in and subsequently verified by a series of clinical translational studies. 3-5 It is remarkable that all of the clinical trials with anti-cgrp medications have been positive, whether in targeting acute attacks, preventing attack onset, or reducing attacks in chronic migraine or frequent episodic migraine. 6,7 In addition, these effects have been achieved without significant adverse events. The CGRP-related treatments do not cause vasoconstriction, one of the major limitations in the use of triptans. CGRP receptor antagonists, the gepants, were shown some time ago to be effective in acute migraine; however, their clinical development was temporarily halted due to liver toxicity following continuous exposure to the initial drugs. Currently, ubrogepant, which is putatively without the toxicity issue, is now in phase III trials. In addition to the Conflict of Interest: The author declares no support from any organization for the submitted work. 47

2 48 May 2017 gepants, a series of fully humanized monoclonal antibodies against CGRP or the CGRP receptor have been developed for prophylactic treatment of chronic migraine (attacks >15 days/month) and for frequent episodic migraine. The antibodies currently in clinical trials are showing significant relief in chronic and frequent episodic migraine with no serious side effects. The site of action of anti-migraine drugs has been discussed for decades. Accumulating evidence suggests migraine is initiated in subcortical brain regions, as exemplified by premonitory studies 8 and continuous scanning of the migraine cycle. 9 Current work suggests that a putative driver of migraine pathology lies in the hypothalamo-brainstem connectivity, hypothalamic links with the spinal trigeminal nuclei to the trigeminovascular system, as well as to the migraine generator in the dorsal rostral pons. 9,10 Identification of these key regions and connections is intriguing, but still very little is known about the underlying molecular mechanisms in migraine. Imaging studies show activation of regions that contain numerous neurons and nerve fibers, with a vast array of neurotransmitters, connected with billions of synapses and receptors. This complexity underscores the difficulty in pinpointing putative therapeutic targets within these regions. CGRP does play a role in a number of the central brain regions associated with migraine; however, it seems unlikely that these sites are the direct targets of the effective CGRP therapies. Gepants are small molecules with limited ability (2%) to cross the blood brain barrier (BBB), and the antibodies are much larger molecules (1500 times larger) that are unable to pass the BBB (<0.01%), thus effectively excluding them from having a major site of action within the CNS. This suggests that the targets of the anti-migraine agents are located in areas not limited by the BBB, such as intra- and extracranial blood vessels, dural mast cells, and peripheral parts of the trigeminal system. In this article I will briefly touch on the history behind the discovery of the role of CGRP in migraine and discuss current aspects of the complexity of the trigeminovascular pathway and the messenger molecules involved. Innervation of Cranial Tissues Related to Migraine. While the cranial autonomic and sensory systems have been studied for decades, the signalling molecules involved were only known indirectly. In the early studies, it was generally assumed that each nerve cell synthetizes and releases only one type of neurotransmitter, a concept known as Dale s principle. 11 This was exemplified by the sympathetic system with noradrenaline and the parasympathetic nerves with acetylcholine, both of which innervate the cerebral arteries and other cranial structures. Demonstration of vesicular noradrenaline in perivascular nerve networks in cerebral arteries was first possible with the Falck-Hillarp histofluorescence method, 12 while the parasympathetic nerves were identified using indirect methods such as the acetylcholinesterase staining method. 13 These autonomic nerve fibers showed close ultrastructural associations at the level of the perivascular nerve terminals and exhibited functional interactions. 14 The classical paradigm, however, was challenged by findings that ATP and adenosine also can be released with catecholamines, both in the periphery (adrenal medulla) and in the brain. 15 A paradigm shift occurred with the development of immunohistochemical methods that could be used to demonstrate neuropeptides. In the cranial circulation, this was first exemplified by the demonstration of vasoactive intestinal peptide (VIP) in parasympathetic nerve fibers of the cerebral circulation. 16,17 Subsequently, several regulatory peptides were found co-localized with classical neurotransmitters. 18 The technological advancements resulted in a decade of intense research in which numerous neuropeptides were demonstrated in various parts of the body. During this period, our interest focused on the brain circulation and the role of neuropeptides in particular. 19 We developed methods to understand the role of these peptides in regulation of the cerebral circulation using combined anatomical, neurochemical, and functional approaches. The autonomic and sensory systems were soon found to contain numerous neuronal messenger molecules, yet even now, their functional significance is only partially understood (Fig. 1).

3 Headache 49 Fig. 1. Schematic demonstration of the perivascular nerves in intracranial arteries. (i). The sympathetic fibers originate in the superior cervical ganglion and store noradrenaline (NA), ATP, and neuropeptide Y (NPY). (ii). Parasympathetic nerves have their origin mainly in the otic and sphenopalatine ganglia and store acetylcholine (ACh), vasoactive intestinal peptide (VIP), peptide histidine isoleucine or methionine (PHI/PHM), pituitary adenylate cyclase activating peptide (PACAP), nitric oxide synthase (NOS). In addition, some data suggest the presence of helodermin, galanin, and gastrin releasing peptide (GRP). (iii). The sensory fibers to the intracranial vasculature have their origin in the trigeminal ganglion and store calcitonin generelated peptide (CGRP), amylin, substance P, neurokinin A and B, PACAP, dynophine, and nociception. The Complexity of the CGRP Family of Peptides and Their Associated Receptors. Calcitonin (CT) is a well-known peptide hormone, isolated from parathyroid glands and shown to be involved in calcium ion homeostasis. Early studies demonstrated a weak anti-nociceptive effect of salmon CT in pain. A breakthrough in this field came after cloning of the CT gene: the gene showed alternative RNAprocessing in neuronal tissues resulting in mrna for a related polypeptide named calcitonin generelated peptide (CGRP). 20 CGRP appears to be the principle gene product formed in tissues outside the thyroid 21 and it coexists with substance P in sensory nerves and neurons. 18,22 The CGRP family of peptides consists not only of CGRP but includes calcitonin (CT), adrenomedullin (AM), and amylin (AMY), 23 and these peptides have been found in the trigeminal system and to some extent in the CNS. The receptors for members of the CGRP peptide family are somewhat unusual in that they consist of the calcitonin receptor-like receptor (CLR), a G protein-coupled receptor of the B-type that is linked to one of three receptor activity-modifying proteins (RAMP). Both components, CLR and RAMP, are necessary to yield a functional receptor. 23 The CGRP receptor consists of CLR and RAMP1. The adrenomedullin receptors, AM 1 and

4 50 May 2017 AM 2, consist of CLR coupled with either RAMP2 or RAMP3, respectively. 23 The calcitonin receptor (CTR) consists of only CTR. Amylin receptors are created by linking CTR with a RAMP; amylin AMY 1-3 receptors consist of CTR plus RAMP1, 2, or 3, respectively. CTR with RAMP1 also responds to CGRP. Localization of CGRP and Related Peptides in the Trigeminal Ganglion. The trigeminovascular system is involved in the regulation of the cranial vasculature and is a key element in transmission of pain. Over the years, the trigeminal system has become a key focus of efforts to elucidate primary headache pathophysiology. As pointed out in Figure 1, there are many potential neuronal messenger molecules in the trigeminal ganglion, and considerable effort has been made to understand their relative roles in pain transmission. Initially the focus was on substance P, driven by the hypothesis that migraine is associated with neurogenic inflammation in the dura mater. 24 For decades, substance P was considered to be the key player in pain responses; however, after a number of negative clinical trials, this line of research was abandoned. 25 Early work on CGRP demonstrated that this sensory peptide was present in >50% of the trigeminal neurons and in small to medium sized (C-fibers). In addition, the wide distribution of CGRP receptors in the trigeminovascular system is consistent with a role in migraine pathophysiology (Fig. 2). CGRP and CGRP receptor elements are expressed in trigeminal nerve fibers, both in central and peripheral branches; however, the CGRP positive neurons and the CGRP receptor containing neurons are distinct in the trigeminal ganglion as well as in their ramifications, the C-fibers and A-fibers, respectively. We have demonstrated the presence of mrna and protein for two CGRP-responsive receptors, the CGRP and AMY1 receptors, in neurons of rat and human trigeminal ganglia. 29 In support of this finding, quantification of agonist and antagonist potencies reveals a dual population of functional CGRPresponsive receptors in primary cultures of rat trigeminal neurons. Role of CGRP in the Trigeminovascular Pathway. Cerebrovascular CGRP fibers originate in the trigeminal ganglion, 22 CGRP is a potent vasodilator in different vascular regions, and we showed that this response occurred in cerebral arteries. Vasodilation was independent of the endothelium and was mediated via activation of adenylate cyclase. 30 In vivo CGRP potently dilates cerebral arterioles, but not cerebral veins, thereby increasing cerebral blood flow. It is now clear that CGRP is an essential molecule responsible for maintaining normal resting tone in the brain circulation. 31 Perivascular CGRP disappears after denervation of the trigeminal ganglion; but this procedure does not alter resting cerebral blood flow, flow-metabolism coupling, nor does it modify cerebral autoregulation. 31 Instead, the CGRP innervation appears to mediate a protective vasodilatory reflex triggered in response to vasoconstriction. 32 Our translational work in humans demonstrated that stimulation of the trigeminal pathway (in neuralgia patients) caused release of both CGRP and substance P. 33 In a landmark clinical study by Edvinsson and Goadsby, it was shown that only CGRP is released in significant amounts during acute migraine and cluster headache attacks. 3,4,34 This study was possible at the time because we had developed a sensitive radioimmunoassay (RIA) for CGRP and also realized that the samples must be taken close to the event (venous outflow from the head the external jugular vein) in order to pick up a significant signal. 33,35 It was astonishing that CGRP was the only peptide/neuronal messenger in the trigeminal system that significantly correlated with the acute attack. 35 Further patient studies supported these findings by demonstrating increased levels of CGRP in serum, cerebrospinal fluid, and saliva. 34,36 Moreover, the elevated levels of CGRP normalize after effective triptan treatment of the migraine attack. 4 In chronic migraine, CGRP seems to be chronically elevated. 36 The systemic administration of CGRP to migraine sufferers triggers a migraine-like attack phenotypically similar to the subject s spontaneous attack. 37 Studies like these paved the way for development of new migraine drugs aimed at various aspects of the CGRP transmission.

5 Headache 51 Fig. 2. Schematic illustration of some of the intracranial sites where the novel CGRP antibodies, the CGRP receptor antibody, and gepants putatively have their main site of action: (i). The trigeminal ganglion with neurons and nerve fibers, satellite glial cells, and blood vessels all have components of the CGRP system. (ii). Dura mater with the meningeal artery and its branches. (iii). Extracranial structures lack blood brain barrier, which allows these agents to interact with the CGRP signaling at many sites. The central part of the trigeminovascular system, the trigeminal nucleus caudalis, and the spinal cord at C 1 and C 2 levels might also be modified by the antibodies and gepants; however, this would more likely be an indirect effect. CGRP Peptides in Migraine-Associated CNS Regions. It has been hypothesized that CGRP acts at second order neurons in the trigeminal nucleus caudalis (TNC) and at the C 1-2 level of the spinal cord to transmit pain signals to the thalamus and higher cortical pain regions. 38 Also of interest are certain brainstem areas that are shown with functional imaging to be activated during migraine attacks. 1,9,10 To study the possible role of CGRP in the CNS, we used in situ hybridization and immunofluorescence to detect mrna expression and cellular localization of CLR and RAMP1, respectively. To define CGRP receptor binding sites, in vitro autoradiography was performed with a labelled CGRP receptor antagonist. CLR and RAMP1 mrna and protein expression were detected in a number of relevant regions, such as periaqueductal gray, area postrema, pontine raphe nucleus, spinal trigeminal nucleus, and spinal cord. 39 In addition, RAMP1 mrna expression was found in the posterior hypothalamus, dorsal raphe nucleus, pontine nuclei, vagus nerve, inferior olive, and motor trigeminal nucleus. Protein coexpression of CLR and RAMP1 was also observed. The

6 52 May 2017 findings suggest that several regions in the brainstem may be involved in CGRP signaling. 39 The distribution within the CNS of CGRP, CLR, and RAMP1, however, is not uniform. CGRP appears most prominently expressed in the soma of neurons in cortex and cerebellum but there is a scant distribution of CGRP positive fibers in the CNS. CLR and RAMP1 immunoreactivity appear mostly in extensive networks of fibers. 40 Overall, the analysis of the distribution of CGRP and its receptor in the CNS shows a very rich expression; hence numerous possibilities may exist for involvement of this neuropeptide in brain function in general. 39,40 We have recently revealed the complexity in the CNS of the different elements of the CGRP/CT system and indeed there is an impressive differential expression of the receptor components in the CNS. 39,40 The finding of a functional noncanonical CGRP receptor (AMY1) at neural sites, suggests this target could be of importance for treating craniofacial pain and also provide another way to target the CGRP axis in migraine. 29 We observed CTR expression in the human brain stem, specifically the medulla oblongata. A dense CTR staining was noted in several discrete nuclei, such as the nucleus of the solitary tract, hypoglossal nucleus, cuneate nucleus, spinal trigeminal nucleus, gracile nucleus, and the inferior olivary nucleus. In addition, we found CTR staining in the area postrema, the lateral reticular nucleus, and the pyramidal tract. Thus, the extensive expression of CTR in the medulla suggests that CTR may be involved in a wide range of CNS functions. 41 It is likely that all the members of the CGRP family of peptides will be found as well as their specific receptor subtypes in related CNS regions. As yet, however, the expression pattern is difficult to interpret in terms of headache and headache treatment. Where Do the CGRP Blockers Act?. Convincing evidence for the role of CGRP in migraine pain came from the development of CGRP receptor antagonists 42 and their subsequent study in clinical trials. 6,7,34 It has been suggested that elevated neuronal RAMP1 could potentially sensitize the trigeminal ganglion of individuals to CGRP actions. However, little is currently known about the regulation of RAMP1 levels in migraine. Published clinical studies using CGRP receptor antagonists have all demonstrated clinical efficacy comparable to that of triptans in the treatment of acute migraine attacks 6,7,34 ; therefore, it is of great importance to clarify the sites where drugs blocking CGRP signaling may have their therapeutic effect. Because of the BBB and the fact that the size of the anti-cgrp and anti-cgrp receptor antibodies are very large, the site of their antimigraine effect is probably outside the BBB. 43 We base this view on several studies: First, autoradiography showed CGRP binding sites in the trigeminal ganglion of rhesus monkey, and this correlated with localization of CLR/RAMP1. 44 Systemic administration of Evans blue revealed that the trigeminal ganglion is not protected by the BBB. This suggests that molecules do not need to be CNS-penetrant to block the CGRP receptors. 44 Second, a PET study in monkey and man using a PET tracer (MK-4232), which displays rapid brain uptake, showed a characteristic regional brain binding at CGRP receptors. 45 In a clinically relevant dose of telcagepant (140 mg, p.o.) there was no displacement seen in the PET binding of radiolabeled tracer in the CNS. At a nearly 10 times higher systemic dose of telcagepant, there was significant displacement of the CGRP receptor binding in the CNS but at no additional clinical effect. The main conclusion was that it is unlikely that antagonism of central CGRP receptors is required for the efficacy of CGRP blockers in acute attacks of migraine. Third, direct measurements of the permeability surface area (PS product 5 ll/min/g tissue) showed >30 times higher PS product in the TG than in any brain region, including the trigeminal nucleus caudalis. 46 Summarizing the current knowledge, one can conclude that (i) the trigeminal ganglion and the dura mater lack BBB, (ii) the trigeminal ganglion contains receptors for triptans and gepants, (iii) this ganglion is a key region for transmission of pain information, eg, to the TNC, and (iv) it innervates cranial vasculature that responds to various migraine related drugs. Therefore, the trigeminal ganglion represents a key structure located strategically at the base of the brain and central to sensory pain

7 Headache 53 Fig. 3. Description of the synaptic localization of CGRP receptors and targets for antimigraine effects of triptans, gepants, and antibodies. Current view suggests that (i) the triptan receptors (5-HT 1B / 1D ) are presynaptic and inhibit the release of CGRP. (ii) The gepants are competitive receptor antagonists at the postsynaptic CGRP receptor and thereby limit is effects. (iii) The CGRP receptor antibody binds to the two extracellular domains of the CGRP receptor, CLR and RAMP1, irreversibly, and thereby reduces synaptic transmission signaling. (iv) The CGRP antibodies act like as sink in the signaling and bind CGRP molecules wherever they find these molecules; they do not discriminate between a- or b-cgrp. neurotransmission. It consists of neurons of various sizes (10%) and a large population of satellite glial cells (90%). In addition, there are CGRP containing nerve fibers within the trigeminal ganglion, providing a morphological basis for interaction between the different cells in the ganglion (Fig. 3). Considering all of the above, CGRP is established to be the key player in understanding migraine pathophysiology. Furthermore, it appears evident that the targets of CGRP reside outside the BBB. With this current knowledge, the drive to fully understand the development of migraine should focus on these structures, with particular focus on the common denominator: the trigeminal ganglion. STATEMENT OF AUTHORSHIP The author conceived and wrote the manuscript in all aspects. REFERENCES 1. Goadsby PJ, Holland PR, Martins-Oliveira M, et al. Pathophysiology of migraine: A disorder of sensory processing. Physiol Rev. 2017;97:

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