Calcitonin receptor antibody validation and expression in the rodent brain

Abstract

Background and aim Therapeutics that reduce calcitonin gene-related peptide activity are effective migraine treatments. However, gaps remain in our understanding of the molecular mechanisms that link calcitonin gene-related peptide to migraine. The amylin 1 receptor responds potently to calcitonin gene-related peptide, and to the related peptide amylin, but its role in relation to either peptide or to migraine is unclear. We sought to better understand the expression of the amylin 1 receptor protein subunit, the calcitonin receptor, in the rodent brain. Methods We profiled three antibodies for immunodetection of calcitonin receptor, using immunocytochemistry, western blotting, and calcitonin receptor conditional knockout mouse tissue. Selected migraine-relevant rat brain regions were then examined for calcitonin receptor-like immunoreactivity. Results All three antibodies detected calcitonin receptor protein but only one (188/10) produced robust immunostaining in rodent brain, under the conditions used. Calcitonin receptor-like immunoreactivity was apparent in the rat brainstem and midbrain including the locus coeruleus, periaqueductal grey and spinal trigeminal nucleus. Conclusions Anti-calcitonin receptor antibodies require comprehensive profiling to ensure confidence in the detection of calcitonin receptor. Using a validated antibody, calcitonin receptor-like immunoreactivity was detected in several brain regions relevant to migraine. Further research is needed to understand the functional consequences of calcitonin receptor expression for calcitonin gene-related peptide or amylin physiology and pathophysiology.

CGRP and the Calcitonin Receptor are Co-Expressed in Mouse, Rat and Human Trigeminal Ganglia Neurons

“…For CTR, we used pAb188 for these experiments. pAb188 has been knockout validated in several studies and displays robust immunoreactivity in several regions in rodent nervous tissue ( Goda et al, 2018 ; Coester et al, 2020 ; Hendrikse et al, 2022 ).…”

“…The predicted molecular weight of the rat and mouse CT (a) splice variant is approximately 52–53 kDa, although the CTR can also exist as other molecular forms due to alternative splicing, dimerization and post-translational modifications ( Albrandt et al, 1995 ; Quiza et al, 1997 ; Harikumar et al, 2010 ). Rodent CTR protein expression was profiled using the monoclonal anti-rat CTR antibody mAb8B9, which has been well-validated and is suitable for western blotting ( Hendrikse et al, 2022 ). In addition, the polyclonal anti-rat CTR antibody pAb188 was also used to allow a direct parallel to the immunohistochemistry data, although this antibody has been previously shown to be less suitable for western blotting ( Hendrikse et al, 2022 ).…”

“…Rodent CTR protein expression was profiled using the monoclonal anti-rat CTR antibody mAb8B9, which has been well-validated and is suitable for western blotting ( Hendrikse et al, 2022 ). In addition, the polyclonal anti-rat CTR antibody pAb188 was also used to allow a direct parallel to the immunohistochemistry data, although this antibody has been previously shown to be less suitable for western blotting ( Hendrikse et al, 2022 ).…”

“…Preparation of whole cell or whole tissue lysates and western blotting were performed as previously described ( Hendrikse et al, 2022 ). Transfected HEK293S cells in 15 cm 2 dishes were washed with ice-cold PBS.…”

“…Measures of protein expression with validated antibodies are therefore important. Previous immunohistochemical studies localizing CTR in the TG used antibodies that were uncharacterized or later found to be unable to detect CTR in a particular species ( Walker et al, 2015 ; Edvinsson L. et al, 2020 ; Hendrikse et al, 2022 ). Therefore, it is necessary to conduct additional studies with well-validated, species-appropriate antibodies to substantiate these prior reports.…”