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 near 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 knock out (KO) animals.
We applied immunohistochemistry (IHC) on 15 TGs from three different genotypes of mice (wild type, αCGRP heterozygotes 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. Moreover, bioinformatical analyses of the primary, secondary, and tertiary structure of both mouse and human isoforms were conducted.
The IHC, evidently 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 report successfully knocks out αCGRP, which is the most dominant isoform present in the TG. 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. Although there were notable differences, similar bioinformatic variations were observed between the two human isoforms.
Our data suggests that only αCGRP and not βCGRP locate within the axons of the mouse TG neurons. Furthermore, the isoforms appear to be sorted differentially into secretory vesicles in the cell bodies of TG neurons.