Amylin, CGRP and the AMY1 receptor: Localisation in the trigeminal ganglia and receptor regulation

Abstract

Calcitonin gene-related peptide (CGRP) is a neuromodulatory peptide that is highly expressed in the nervous system. It is involved in several physiological and pathophysiological processes, including pain and migraine. CGRP is closely related to amylin, an endocrine hormone secreted from the pancreas in response to food intake with a role in metabolism. CGRP and amylin mediate their actions via class B G protein-coupled receptors. Amylin can potently activate three amylin receptors, formed by the association of the calcitonin receptor (CTR) and one of three receptor activity-modifying proteins (RAMPs). CGRP can potently activate its canonical CGRP receptor and the amylin 1 (AMY1) receptor, comprised of CTR and RAMP1. To add further complexity, the CTR can be alternatively spliced, generating variants that differ in their pharmacology and signalling profiles. CGRP and the CGRP receptor have well described roles in migraine pathogenesis, resulting in the generation of breakthrough treatments targeting their activity. However, many patients do not respond to these therapeutics, representing an unmet clinical need and signifying that further druggable targets are required. Recently, amylin and the AMY1 receptor have been identified as such targets and are reportedly expressed in the trigeminal ganglia (TG), an important structure involved in migraine. However, the presence of amylin and CTR in the TG is not well defined, due to poorly characterised tools and limited translation across species. Similarly, the regulatory behaviour of the AMY1 receptor remains largely unknown. Therefore, this thesis aimed to explore the relative distribution of amylin, CGRP and the AMY1 receptor in the TG of multiple species and compare the regulatory profiles of the AMY1 and CGRP receptors. The experimental strategies chosen to investigate protein expression was immunohistochemistry and immunoblotting. Therefore, a major component of this thesis was characterising antibodies for specificity and selectivity, to determine whether the observed immunoreactivity was genuine. Chapter Three and Chapter Four investigated the relative distribution of amylin, CGRP and the AMY1 receptor component, CTR, in the TG of mice, rats, and humans using thoroughly validated antibodies. Chapter Five pharmacologically characterised four fluorescently labelled agonists of the CGRP and AMY1 receptors, which were then used in Chapter Six to examine the internalisation profiles of these two receptors. Antibodies specific for CGRP, amylin and CTR were identified and used to investigate expression. CGRP was highly expressed the TG. Conversely, amylin-like immunoreactivity indicated that little or no amylin was present. When the spatial relationship between the CTR and CGRP was explored, colocalisation was frequently seen in small to medium C-fibre neurons. Additionally, western blotting indicated the presence of CTR splice variants across species. The addition of a fluorescent label to the agonists was generally well tolerated at the CGRP and AMY1 receptors. Differences in receptor internalisation were identified between the two CGRP-responsive receptors, suggesting they could mediate different aspects of CGRP’s biology. This thesis offers an expanded perspective on the expression and activity of the AMY1 receptor and its endogenous peptides in the TG, which is critical for advancing the understanding of these peptides’ biology and the development of therapeutics.