Exploring expression of amylin and CGRP receptors in the rat brain

Abstract

The calcitonin family of peptides modulate a range of physiological processes that make them amenable to therapeutic exploitation. The neuropeptide calcitonin gene-related peptide (CGRP) has particular value as it plays a major role in the pathophysiology of migraine. The related hormone amylin is also therapeutically relevant for both diabetes and obesity due to its effects on satiation and glucoregulation. However, both CGRP- and amylin-based treatments have limitations including modest efficacy. Improving the clinical value of these two peptides is, in part, restricted by incomplete understanding of the molecular pathways through which they mediate specific (patho)physiological processes. CGRP and amylin have sites of action in the brain, where radioligand binding reveals widespread binding sites for both peptides. The molecular correlates responsible for these binding sites, which represent cellular receptors, are not yet known. The in vitro pharmacology of CGRP and amylin receptors is complex due to their unusual heterodimeric nature. The canonical receptor for CGRP comprises a G protein-coupled receptor, known as the calcitonin-like receptor (CLR), coupled to a receptor activity-modifying protein (RAMP1). Interestingly, while the related calcitonin receptor (CTR) complexed with RAMP1 is termed an amylin 1 (AMY1) receptor, it is potently activated by both CGRP and amylin in vitro. It is not clear if the AMY1 receptor is a genuine receptor for both peptides in vivo. A foundational step in understanding the role of the AMY1 receptor in CGRP and amylin biology is clearly defining the expression patterns of the two receptor components (CTR and RAMP1) in the brain, where both peptides are active. Where it is available, expression information is often incomplete. Therefore, the aim of this thesis was to examine the tissue localisation of CTR and RAMP1 in rat brain. Select regions of the brain were targeted based on a comprehensive literature review including peptide binding sites, RNA and protein expression and physiological relevance. The experimental strategy chosen was immunohistochemistry, which uses antibodies as specific probes. Therefore, an important first step was characterising the antibodies chosen for their selectivity and suitability, using complementary cell-based methods. In the rat brain, robust CTR-like immunoreactivity was observed in many regions relevant for CGRP and amylin, particularly in the brainstem. RAMP1-like immunoreactivity was also robust but varied between antibodies. Ultimately, transgenic mouse models revealed that RAMP1-like immunoreactivity could not be confidently attributed to the presence of RAMP1. This has implications for previous studies and our overall understanding of RAMP1 expression in the brain. This thesis offers an expanded perspective on the receptors for CGRP and amylin in the brain, which is critical for advancing the understanding of these peptides’ biology as well as their clinical utility.