A Tale of Two Peptides: Identifying Targets for Migraine

Abstract

Calcitonin gene related-peptide (CGRP) and amylin are closely-related peptides that are both reported to be elevated in the plasma of migraine sufferers. CGRP is highly expressed in neural tissues and migraine relevant structures, including the trigeminal ganglia (TG). Amylin is secreted from pancreatic islets in response to food intake, with conflicting reports of expression in the TG. CGRP potently activates two receptors, the CGRP receptor and Amylin 1 (AMY1) receptor, whereas amylin potently activates the AMY1 receptor. CGRP has been proposed to be auto regulated in an autocrine manner in the TG, however CGRP does not colocalise with the CGRP receptor in the TG. The AMY1 receptor is also expressed in the TG, but where it is expressed and whether it is found together with CGRP or amylin is poorly understood. Therefore, our aim was to determine whether amylin is expressed in the TG and the relative distribution of AMY1 receptor components with CGRP or amylin using thoroughly validated antibodies. To study the distribution of CGRP, amylin and the AMY1 receptor component, calcitonin receptor (CTR), in the TG a range of antibodies were validated. Anti-CGRP and amylin antibodies were validated using immunoblotting and rat pancreas histology. Anti-CTR antibodies were validated using transfected HEK293S cells and knock out tissue. Lead antibodies were then applied to mouse, rat and human TG sections with neural markers to investigate their distribution. Anti-amylin antibodies frequently cross-reacted with CGRP, whereas anti-CGRP antibodies displayed no cross-reactivity with amylin. Lead anti-amylin antibodies displayed little or no detection of amylin in rodent and human β-tubulin III-positive neurons, respectively, whereas abundant CGRP-like immunoreactivity was observed across all species. When co-incubated the little amylin-like immunoreactivity observed strongly colocalised with anti-CGRP staining. This was particularly apparently with T4145, the more cross-reactive amylin antibody. The lead anti-CTR antibody, 188/10, displayed robust detection of rodent CTR in transiently transfected HEK293S cells and a marked loss of staining in knock out mouse nucleus accumbens compared to wild type. CTR-like and CGRP-like immunoreactivity frequently colocalised in rat TG sections, primarily in C-fibre neurons, and rarely in NF200-positive A-fibre neurons. These results demonstrate the importance of thoroughly validating antibodies. The data suggest that in the TG, CGRP, rather than amylin, is the endogenous agonist for the AMY1 receptor. Additionally, it suggests that CGRP autocrine signalling occurs via the AMY1 receptor.