18th European Headache Congress

Professor

Chair

Are amylin receptors relevant in migraine?

Invited speaker

Invited lecture

Central effects of CGRP: focus on the cerebellum

Peripheral effects of CGRP: focus on the trigeminal ganglion

Pharmacological characterization of ubrogepant and atogepant in cAMP assays at human, rat and mouse calcitonin family receptors in transfected cells

CGRP (receptor) inhibitors in the clinic

Ubrogepant and atogepant belong to the "gepant" class of CGRP receptor antagonists. There are limited data on their ability to antagonize different members of the wider calcitonin receptor family. This study aimed to address this by comparing antagonism by ubrogepant and atogepant at the CGRP, calcitonin, adrenomedullin, and amylin receptors from three species.Cos7 cells transiently transfected with human, rat or mouse receptor subunits for all seven calcitonin family receptors were exposed to species-matched agonists in the absence or presence of ubrogepant and atogepant. Two other gepants (rimegepant and telcagepant) were also studied as comparators in some experiments. As all of these receptors robustly couple to the Gαs G protein, cAMP production was quantified and antagonist potency calculated.Ubrogepant and atogepant were antagonists of the human CGRP receptor and were also able to antagonize the AMY1(a) receptor. Selectivity estimates between these two human receptors varied depending on assay format and agonist used. As an example of the results, Table 1 shows IC50 values at the human CGRP and AMY1(a) receptors. Antagonism was weaker at mouse and rat receptors, compared to human. Depending on the species, antagonism of other receptors was sometimes observed. Overall, there were some differences in selectivity between receptors in the different species.Ubrogepant and atogepant are capable of antagonizing additional receptors in the calcitonin receptor family, beyond the canonical CGRP receptor.

Abstract lecture

In-depth analysis of CGRP Isoforms: only αCGRP localizes in Axons of the mouse Trigeminal Ganglion

Migraine

The neuropeptide calcitonin gene related peptide (CGRP) has been established to be a key signaling molecule in migraine, but little is known about the differences between the two isoforms: αCGRP and βCGRP. Previous studies have been hampered by their close similarity, making the development of specific antibodies near impossible. In this study we sought to test the hypothesis that αCGRP and βCGRP localize differently within the neurons of the mouse trigeminal ganglion (TG), using knock out (KO) animals.We applied immunohistochemistry (IHC) on 15 TGs from three different genotypes of mice (wild type, αCGRP heterozygotes and αCGRP KOs) with a primary antibody targeting the mature neuropeptide sequence of both αCGRP and βCGRP. Subsequently, the localization patterns of the two isoforms were analyzed. Moreover, bioinformatical analyses of the primary, secondary, and tertiary structure of both mouse and human isoforms were conducted.The IHC, evidently showed that the key isoform localized within the axons of the mouse TG neurons, is αCGRP and not βCGRP. Furthermore, differences in intensities indicate that the model used in this report successfully knocks out αCGRP, which is the most dominant isoform present in the TG. We further categorized the localization patterns of CGRP in neuronal cell bodies in the TG and found using bioinformatic analyses that differences in localization might be explained by intracellular peptide sorting. Although there were notable differences, similar bioinformatic variations were observed between the two human isoforms.Our data suggests that only αCGRP and not βCGRP locate within the axons of the mouse TG neurons. Furthermore, the isoforms appear to be sorted differentially into secretory vesicles in the cell bodies of TG neurons.

αCGRP and βCGRP are co-expressed in the trigeminal ganglia

Preclinical research on novel pharmacological targets

Calcitonin gene-related peptide (CGRP) is a neuropeptide involved in pain transmission and migraine pathogenesis. CGRP has two major isoforms, αCGRP and βCGRP, which differ by three amino acids in humans and mice. αCGRP is highly expressed in the trigeminal ganglia (TG) and has a well-established role in pain sensitisation and migraine. In contrast, βCGRP expression is poorly defined and is often reported to be primarily expressed in the enteric nervous system, with limited relevance to migraine. Therefore, this study aimed to compare the relative distribution of αCGRP and βCGRP in the mouse TG.Detection of the mouse CGRP isoforms by anti-CGRP antibodies was tested by dot blot. Wildtype (WT) and αCGRP knockout (KO) adult mouse TG (C57BL/6J background, WT 3M/1F, KO 4F) were immunostained for CGRP relative to neuronal markers (β tubulin III and NF200) and the calcitonin receptor (CTR). RNA-fluorescent in situ hybridisation (RNA-FISH) probes against αCGRP (Calca) and βCGRP (Calcb) mRNA were validated in transfected HEK293S cells. These probes were then used to examine isoform expression in WT adult mouse TG (C57BL/6J, 2F/3M).All anti-CGRP antibodies tested detected both isoforms of CGRP. CGRP-like immunoreactivity was present in WT and αCGRP KO mouse TG in neuronal cell bodies, often colocalising with CTR. However, compared to the WT mice, CGRP immunoreactivity in the αCGRP KO TG was of a lower fluorescence intensity and lacked pearl-like fibre immunoreactivity. The RNA-FISH probes were specific to each isoform and indicated that αCGRP and βCGRP mRNA were often present in the same neuronal cell bodies, although some neuronal cell bodies only expressed one CGRP isoform.βCGRP is expressed in mouse TG neurons, suggesting it plays a role in neuromodulation and nociception, alongside αCGRP. Both isoforms may be important for mediating CGRP's pathogenic activity in migraine.

Plenary hall

CGRP and its receptors – What's new? 

Parallel session

CGRP and its receptors – What's new?

Appointment

Chair

Programme