Differentially localizing isoforms of the migraine component calcitonin gene-related peptide (CGRP), in the mouse trigeminal ganglion: βCGRP is translated but, unlike αCGRP, not sorted into axons.

Abstract

Objective: 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 nearly 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 αCGRP knock out (KO) animals.

Methods: We applied immunohistochemistry (IHC) on 15 TGs from three different genotypes of mice; wild type (WT) αCGRP heterozygote (Het) 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. Furthermore, similar IHCs were produced in KO animals after being treated with monoclonal CGRP antibodies to study the origin of the observed CGRP. Additional IHCs were conducted in KO and WT mice to locate CGRP sorting peptides within neuronal cell bodies. Lastly, bioinformatical analyses of the primary, secondary, and tertiary structure of the two isoforms were conducted.

Results: The IHC 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 study successfully knocks out αCGRP. 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. IHC following injections with monoclonal CGRP antibodies in KO mice ruled out the possibility that the βCGRP observed in trigeminal neurons had peripheral origins. This conclusion was enhanced by IHC experiments which showed the presence of CGRP co-localizing sorting peptides in KO mice.

Conclusion: Our data show that mainly αCGRP and not βCGRP locate within the axons of the mouse TG neurons. The βCGRP observed within the TG neuronal cell bodies is synthesized intracellularly and not taken up from the environment. Furthermore, the isoforms appear to be sorted differentially into secretory vesicles in the cell bodies of TG neurons.